Apparatus for assay, synthesis and storage, and methods of manufacture, use, and manipulation thereof

ABSTRACT

The invention features methods of making devices, or “platens”, having a high-density array of through-holes, as well as methods of cleaning and refurbishing the surfaces of the platens. The invention further features methods of making high-density arrays of chemical, biochemical, and biological compounds, having many advantages over conventional, lower-density arrays. The invention includes methods by which many physical, chemical or biological transformations can be implemented in serial or in parallel within each addressable through-hole of the devices. Additionally, the invention includes methods of analyzing the contents of the array, including assaying of physical properties of the samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/707,501, which was filed Aug. 11, 2005; and is a continuation-in-partof U.S. patent application Ser. No. 10/315,832, which was filed on Dec.10, 2002, which is a divisional application of U.S. patent applicationSer. No. 09/975,496, which was filed on Oct. 10, 2001, and is now issuedas U.S. Pat. No. 6,716,629, and which claims the benefit of U.S.Provisional Application No. 60/239,538, filed Oct. 10, 2000, U.S.Provisional Application No. 60/268,894, filed Feb. 14, 2001, and U.S.Provisional Application No. 60/284,710, filed Apr. 18, 2001; each of theforegoing applications are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention relates to devices for molecular synthesis, storage andscreening, and other chemical, biochemical, biological, and physicalexperiments, and to methods of making, using, and manipulating the same.

BACKGROUND OF THE INVENTION

High throughput methods for creating and analyzing chemical andbiochemical diversity play a vital role in technologies including drugdiscovery and development. Specific applications of high throughputmethods include drug discovery, optimization of reaction conditions(e.g., conditions suitable for protein crystallization), genomics,proteomics, genotyping, polymorphism analysis, examination of RNAexpression profiles in cells or tissues, sequencing by hybridization,and recombinant enzyme discovery.

Rapid, high throughput methods for synthesizing (e.g., usingcombinatorial chemistry methods) and screening large numbers of thesecompounds for biological and physicochemical properties are desired, forexample, to increase the speed of discovery and optimization of drugleads.

Similarly, due in part to the large amount of sequence data from thehuman genome project, efforts are underway to rapidly obtain x-raycrystallography data for the protein products of many newly discoveredgenes. One of the rate limiting steps in this process is the search forappropriate solution conditions (e.g., pH, salt concentration) to causeprotein crystallization. There is also a need to determine the functionof each of the newly discovered genes (i.e., “functional genomics”) andto map protein-protein interactions (i.e., “proteomics”). Given thelarge number of human genes, protein modifications, and protein bindingpartners, higher throughput methods are desired.

Another advance in biotechnology is the creation of surfaces withhigh-density arrays of biopolymers such as oligonucleotides or peptides.High-density oligonucleotide arrays are used, for example, ingenotyping, polymorphism analysis, examination of RNA expressionprofiles in cells or tissues, and hybridization-based sequencing methodsas described, for example, in U.S. Pat. Nos. 5,492,806, 5,525,464, and5,667,972 to Hyseq, Inc. Arrays containing a greater number of probesthan currently provided are desirable.

The process of discovering and improving recombinant enzymes forindustrial or consumer use has emerged as an important economic activityin recent years. A desire to discover very rare, activity-improvingmutations has further stimulated the search for higher throughputscreening methods. Such methods often require screening 100,000 to1,000,000 members of a genetic library in parallel, and then rapidlydetecting and isolating promising members for further analysis andoptimization.

One of the challenges in the development of high throughput methods isthat conventional liquid handling techniques such as pipetting,piezoelectric droplet dispensing, split pin dispensing, andmicrospritzing are generally unsuitable for rapidly loading ortransferring liquids to or from plates of high density (e.g., plateshaving more than about 384 wells). For example, these techniques cancause substantial splashing, resulting, for example, in contamination ofneighboring wells and loss of sample volume. Also, as the number ofwells increases, the time necessary to reformat compounds from theprevious generation of plates to the higher density plates generallyincreases, thus limiting the utility of higher density plates.Evaporation can also be problematic with times greater than a fewseconds. Moreover, entrapped air bubbles can result in inconsistenciesin the loading of small fluid volumes (e.g., less than about onemicroliter).

Significant bottlenecks in high throughput screening efforts includelibrary storage, handling, and shipping. As the number of compounds in alibrary increases, the number of 96- or 384-well plates, and the totalvolume needed to store the libraries, also increases. For compounds thatare stored in frozen solvent such as DMSO or water, thawing, dispensing,and refreezing pose the hazard of crystallization, precipitation, ordegradation of some compounds, making it difficult to dispense accuratequantities in the future. Having samples stored in low-density platesrequires a time consuming step of reformatting the samples intohigh-density plates before the high-density technology can be utilized.

SUMMARY OF THE INVENTION

The invention features methods of making devices, or “platens”, having ahigh-density array of through-holes, as well as methods of cleaning andrefurbishing the surfaces of the platens. The invention further featuresmethods of making high-density arrays of chemical, biochemical, andbiological compounds, having many advantages over conventional,lower-density arrays. The invention includes methods by which manyphysical, chemical or biological transformations can be implemented inserial or in parallel within each addressable through-hole of thedevices. Additionally, the invention includes methods of analyzing thecontents of the array, including assaying of physical properties of thesamples.

In various embodiments, the reagents can be contained within thethrough-holes by capillary action, attached to the walls of thethrough-holes, or attached to or contained within a porous materialinside the through-hole. The porous material can be, for example, a gel,a bead, sintered glass, or particulate matter, or can be the inner wallof a through-hole that has been chemically etched. In particularembodiments, the arrays can include individual molecules, complexes ofmolecules, viruses, cells, groups of cells, pieces of tissue, or smallparticles or beads. The members of the arrays can also, for example,function as transducers that report the presence of an analyte (e.g., byproviding an easily detected signal), or they can function as selectivebinding agents for the retention of analytes of interest. Using thesemethods, arrays corresponding to a large plurality of human genes (e.g.,using nucleic acid probes) can also be prepared.

On embodiment of the invention features a method of making a platen of adesired thickness having a plurality of through-holes. The methodincludes the steps of (a) providing a plurality (e.g., 2, 3, 5, 8, 10,100, 1000 or more) of plates having upper and lower surfaces, whereinone or both of the upper and lower surfaces of at least some of saidplurality of plates has continuous, substantially parallel groovesrunning the length of said surfaces; (b) bonding the upper surfaces ofall but one of said plurality of plates to the lower surfaces of theother plates (i.e., the upper surface of the first plate is bonding tothe lower surface of the second plate; the upper surface of the secondplate is bonded to the lower surface of the third plate; and so on; theupper surface of the last plate is not bonded to anything else); and (c)if necessary to achieve the desired thickness, slicing the platensubstantially perpendicularly to the through-holes, thereby creating aplaten of a desired thickness having a plurality of through-holes. Stepc) can optionally be repeated make a plurality of platens. By “aplurality of through-holes” is meant at least 2 (e.g., 2, 5, 10, 20, 25,50, 100, 200, 250, 500, 25,000, 50,000 or more). For example, a platenthe size of a conventional microscope slide may have about 3,072 holes,while a platen the size of a microtiter plate may have about 24,576. Thenumber of through-holes on a microtiter plate can be 50,000, 100,000,200,000 or more. Of course, the number of through-holes will varydepending on the diameter of the hole and the size of the platen. Forexample, where the through-hole has a diameter of less than about 400micrometers, the through-hole density is at least 1.6 through-holes persquare millimeter.

The plates can be made from any material that can be bonded (e.g.,plastic, metal, glass, or ceramic), and each can have a thickness from,e.g., about 0.01 mm to 2.0 mm, preferably 0.1 mm to 1 mm; the grooveshave a depth from, e.g., 0.005 mm to 2.0 mm (i.e., less than thethickness of the plates); and the grooves can have a width from, e.g.,0.1 mm to 1.0 mm.

The plates can be bonded in a configuration in which the grooves of oneplate are substantially parallel to the grooves of each of the otherplates, or can be bonded so that the grooves of certain plates areperpendicular to, or at acute angles to, the grooves of certain otherplates.

In another embodiment, the invention features a device for theimmobilization of probes, cells, or solvent. The device includes aplaten (optionally having hydrophobic upper and lower surfaces) having aplurality of through-holes (e.g., from the upper surface to the lowersurface), where at least some of the through-holes contain a porousmaterial such as a gel (e.g., polyacrylamide), silica, sintered glass,or polymers for the immobilization of probes, cells, or solvent.

In still another embodiment, the invention features a method of making aplaten having opposing hydrophobic surfaces and a plurality ofhydrophilic through-holes. The method includes the steps of: (a) coatinga plate with a material (e.g., gold, silver, copper, gallium arsenide.metal oxides, or alumina) that reacts with amphiphilic molecules (e.g.,alkane thiols, alkanephosphates, alkane carboxylates); (b) formingthrough-holes in the plate (e.g., by micromachining methods such asdrilling, electrospark discharge machining (EDM), punching, stamping, oretching; and (c) treating (e.g., dipping or spraying) the plate with asolution or vapor of an amphiphilic molecule to provide a platen havinghydrophobic coating on surfaces of the platen but not on the walls ofthe through-holes. The invention also includes the platens made by thismethod, as well as a method of regenerating the hydrophobic coating onthe platen after use. This method includes the steps of (a) removingresidual hydrophobic coating, if any (e.g., by washing the platen withoxidant, reductant, acid, base, or detergent, or by heating,electropolishing, irradiating, or burning); and (b) treating the platenwith a solution or vapor of an amphiphilic molecule to regenerate thehydrophobic coating.

In yet another embodiment, the invention features a method ofselectively making a coating on the surfaces of a platen having aplurality of through-holes. The method includes the steps of: (a)selectively coating the surfaces of the platen with a material thatreacts with amphiphilic molecules; and (b) treating the platen with asolution or vapor of an amphiphilic molecule to regenerate thehydrophobic coating.

Still another embodiment of the invention features a platen having twoopposing surfaces and a plurality of through-holes extending between thesurfaces. The surfaces have different chemical properties relative tothe walls of the through-holes, such that the walls and surfaces can beindependently functionalized. For example, the walls can be coated withgold (e.g., by coating the entire platen, including both the walls andthe opposing surfaces with gold, and then electropolishing the surfacesto remove the gold therefrom), allowing the walls to be renderedhydrophobic upon treatment with alkane thiols. Conversely, the surfaces(but not the walls) could be coated with metal oxides so thatalkanephosphates can be bound thereto.

In another embodiment, the invention features a method of making aplastic platen of a desired thickness, having through-holes. The methodfeatures the steps of: a) potting a plurality of capillaries (e.g.,glass or plastic capillaries) in the through-holes of a stack of platenscomprising at least two platens having through holes; b) separatingadjacent platens by a distance equal to the desired thickness; c)injecting a plastic-forming material into the space between theseparated platens; d) forming (e.g., heat-setting or curing) theplastic; and e) slicing at the interface between the platens and theplastic to form the chips. The plastic-forming material can be, forexample, a photo-, thermo-, or chemical-curable material such as aUV-curable material, e.g., polymethylmethacrylate (PMMA), polystyrene,or epoxy, and the forming step can entail exposing the material toultraviolet light; or the plastic-forming material can be a moltenthermoplastic material and the forming step can involve cooling thematerial.

In still another embodiment, the invention features a method of making aplastic chip of a desired thickness, having through-holes. The methodfeatures the steps of: a) potting a plurality of fibers or wires in thethrough-holes of a stack of platens comprising at least two platenshaving through holes; b) separating adjacent platens by a distance equalto the desired thickness; c) injecting a plastic-forming material intothe space between the separated platens; d) forming the plastic; e)withdrawing the fibers or wires from the plastic to form through-holes;and f) slicing at the interface between the platens and the plastic toform the chips.

Still another embodiment of the invention is a method of creating achemical array. The method includes the steps of: a) providing a platenhaving a plurality of through-holes and two opposing surfaces; b)applying a mask to one or both surfaces of the platen to block at leastsome of the through-holes, while leaving other through-holes open; c)exposing a surface of the platen to a reagent (e.g., e.g., a liquid, agas, a solid, a powder, a gel, a solution, a suspension such as aslurry, a cell culture, a virus preparation, or electromagneticradiation; e.g., by spraying the platen with a solution or suspension ofthe reagent, or by condensing, pouring, depositing, or dipping thereagent onto the platen) so that the reagent enters at least one of theopen through-holes; and d) repeating steps b) and c) (e.g., at leastonce, generally at least three times; for creation of nucleic acidarrays, the steps can be repeated four times the length of the desirednucleic acid chains; for creation of protein arrays, the steps can berepeated twenty times the length of the desired peptide chains) with atleast one different mask and at least one different reagent to create achemical array. The masks can be reusable or disposable, and can beapplied mechanically (e.g., robotically) or manually. The mask can, insome cases, initially include the reagent (e.g., absorbed onto orcontained within it). The mask can be flexible or rigid, for example,and can be made of a polymer, an elastomer, paper, glass, or asemiconductor material. The mask can, for example, include mechanicalvalves, pin arrays (e.g., posts, pistons, tubes, plugs, or pins), or gasjets. In some cases, the “applying” step forms a hermetic seal betweenthe mask and the platen. The mask can also be translated (e.g., movedbetween the repetitions of the method) to expose differentthrough-holes. In some cases, the mask has co-registration pins andholes such that alignment of pins and holes in the mask register withthe through-holes in the platen. In these cases, multiple masks can bemade part of a flexible tape, and the multiple masks are registered withthe through-holes of the platen by advancing the tape (e.g., the maskscan be on a spool, ribbon, or roll, and can be advanced in a manneranalogous to the advancing of film in a camera). Arrays created by anyof these methods are also considered to be an aspect of the invention.

In yet another embodiment, the invention features a method of creating achemical array. The method includes the steps of: a) providing a platenhaving a plurality of through-holes and two opposing surfaces; b)applying a mask that has one or more reagents on its surface to one orboth surfaces of the platen to transfer the reagent from the mask to atleast some of the through-holes; and c) repeating step b) with at leastone different mask and at least one different reagent to create achemical array.

The invention also features a method for separating samples within achemical array in a platen. The method includes the steps of a)providing a platen having a plurality of through-holes and two opposingsurfaces; b) electrophoretically transporting a charged reagent into atleast some of the through-holes by placing the platen into anelectrophoresis apparatus containing the reagent and applying anelectric field parallel to the through-holes; and c) repeating step b)with at least one different reagent to create a chemical array.

In still another embodiment, the invention features a method of creatinga spatially addressable array. The method includes the following steps:a) providing a platen having a spatially addressable plurality ofdiscrete through-holes each having an inner wall, wherein said platenhas opposing hydrophobic surfaces; and b) covalently or non-covalentlyimmobilizing at least one reagent or probe on the inner walls of atleast some of the through-holes or on a bead contained within at leastone of the through-holes to form a spatially addressable array. In thismethod, the through-holes can be either non-communicating (i.e., thecontents of adjacent through-holes do not mix with each other) orselectively communicating (i.e., the walls of at least some of thethrough-holes act as semi-permeable membranes) through-holes. In somecases, the method can also include the step of: c) flowing reagents(e.g., monomers, wash solutions, catalysts, terminators, denaturants,activators, polymers, cells, buffer solutions, luminescent andchromatogenic substrate solutions, beads, heated or cooled liquids orgases, labelled compounds, or reactive organic molecules) into orthrough a predetermined subset of the through holes.

Yet another embodiment of the invention is a method of creating astochastic array. The method includes a) providing a platen having aplurality of through-holes; and b) applying each of a plurality ofreagents to the through-holes in a random or semi-random manner (e.g.,spatially random or random with respect to distribution of reagents) tocreate a stochastic array. The “applying” step can include, for example,providing a plurality of dispensing devices addressing at least some ofthe through-holes, dispensing different combinations of reagentsolutions (e.g., as solutions, neat, or in suspension) into eachthrough-hole, and repositioning the dispensing devices at least once toaddress a different set of through-holes. In this case, the method canalso involve dispensing a fluid that is immiscible with the reagentsolutions into at least one through-hole.

In another embodiment, the invention features a method of identifyingcombinations of reagents having a biological, chemical or physicalproperty of interest. The method involves, for example, the use ofradiolabelled probes, or the measurement of chemiluminescence. Themethod features the steps of: a) creating a stochastic array using theabove method; b) assaying the stochastic array for combinations having aproperty of interest; and c) identifying the reagents that have theproperty of interest. Non-limiting examples of properties of interestinclude catalysis (see, e.g., Weinberg et al., Current Opinion in SolidState & Materials Science, 3:104-110 (1998)); binding affinity for aparticular molecule (see, e.g., Brandts et al., American Laboratory22:3041 (1990); or Weber et al., J. Am. Chem. Soc. 16:2717-2724 (1994));ability to inhibit particular chemical and biochemical reactions;thermal stability (see, e.g., Pantaliano et al., U.S. Pat. Nos.6,036,920 and 6,020,141); luminescence (see, e.g., Danielson et al.,Nature 389:944-948 (1997)); crystal structure (see, e.g., Hindeleh etal., Journal of Materials Science 26:5127-5133 (1991)); crystal growthrate; diastereoselectivity (see, e.g., Burgess et al., Angew. Chem.180:192-194 (1996)); crystal quality or polymorphism; surface tension;(see, e.g., Erbil, J. Phys. Chem. B., 102:9234-9238 (1998)); surfaceenergy (see, e.g., Leslot et al., Phys. Rev. Lett. 65:599-602 (1990));electromagnetic properties (see, e.g., Briceno et al., Science270:273-275 (1995); or Xiang et al., Science 268:1738-1740 (1995));electrochemical properties (see, e.g., Mallouk et al., ExtendedAbstracts; Fuel Cell seminar: Orlando, Fla., 686-689 (1996)); andoptical properties (see, e.g., Levy et al., Advanced Materials 7:120-129(1995)); toxicity, antibiotic activity, binding, and other biologicalproperties; fluorescence and other optical properties; and pH, mass,binding affinity, and other chemical and physical properties.

In another embodiment yet, the invention features a method of loading aplaten having a plurality of through-holes, where the platen hasopposing surfaces (e.g., the surfaces are hydrophobic and thethrough-holes have hydrophilic walls). The method includes the steps of:a) dipping the platen into a liquid sample (e.g., a neat liquid, asolution, a suspensions, or a cell culture) that includes a sample to beloaded into the through-holes, thereby loading at least some of thethrough-holes with the sample; and b) passing the platen through aliquid that has an affinity for the surfaces of the platen but that isimmiscible with the liquid sample, thereby cleaning the surface of theplaten of excess sample mixture (e.g., by adding, on top of the samplemixture, the immiscible liquid, where the liquid has a lower densitythan the sample mixture (e.g., mineral oil); and removing the platenfrom the sample mixture through the liquid; device comprising a barrierbetween the sample and the liquid).

The invention also features another method of loading a platen having aplurality of through-holes, where the platen has opposing surfaces. Themethod includes: a) dipping the platen into a liquid sample comprising asample to be loaded into the through-holes, thereby loading at leastsome of the through-holes with the sample; and b) contacting the platenwith a liquid that has an affinity for the surfaces of the platen but isimmiscible with the liquid sample, thereby cleaning the surface of theplaten of excess sample mixture.

The invention also features a method of maintaining the viability of anaerobic organism in a platen having a plurality of through-holes. Themethod includes the steps of: a) loading the aerobic organism (e.g., acell or an embryo) into at least some of the through-holes of theplaten, and b) submerging the platen into a gas permeable liquid. Theorganism can be, for example, in a fluid such as a growth medium, inwhich case the gas permeable liquid should be immiscible with the fluid.The method can also include assaying one or more physical properties ofthe aerobic organism.

The gas permeable liquid can be, for example, a fluorocarbon such asperfluorodecalin, a silicone polymer, or a monolayer (e.g., a monolayerof a lipid or high molecular weight alcohol.

In another embodiment still, the invention features a method of mixingvolatile samples with other samples (whether volatile or non-volatile).The method include the steps of: a) providing a platen having aplurality of through-holes; b) optionally loading some or all of thethrough-holes with one or more non-volatile samples (if any); c) loadingat least some of the through-holes of the platen with one or morevolatile samples to allow the samples in each through-hole to mix withother samples in the same through-hole; and d) submerging the platen ina liquid immiscible with the volatile samples, where steps b), c) and d)can be performed in any order. In preferred embodiments, step d) isperformed prior to introduction of volatile samples. The samples to bemixed can be initially provided in two separate platens that arecontacted while submerged in said immiscible liquid to allow mixing. Theimmiscible liquid can be, for example, a fluorocarbon, a siliconepolymer, mineral oil, or an alkane.

The invention also features a method of mixing an array of samples. Themethod entails: a) providing a platen having a plurality ofthrough-holes, wherein at least some of the through holes are loadedwith a first sample or set of samples; b) providing a substantially flatsurface comprising an array of a second sample or set of samples,wherein the second sample or set of samples on the flat surface can beregistered (e.g., the second sample or set of samples can be arranged ina spatial pattern that allows it to line up with at least some of thethrough-holes of the platen) with the sample in the platen; c)registering the platen with the array of the second sample or set ofsamples on the flat surface; and d) contacting the platen with the flatsurface, wherein the sample in the platen is aligned with the sample onthe flat surface. This method can be used, for example, to avoidcross-contamination; also, registering and contacting can be donesimultaneously. In some cases, either the first or second sample or setof samples can include one or more probes. The method can also includethe further step of analyzing a physical property (such as fluorescenceor other optical properties, pH, mass, binding affinity; e.g., usingradiolabelled probes and film, chemiluminescence) of a sample containedin the platen. In some cases, the flat surface can also include ahydrophobic pattern matching the pattern of the platen array (e.g., toprevent cross-contamination).

In another embodiment, the invention features a method for transferringa reagent or probe to a receptacle (e.g., into a bottle, a tube, anotherplaten, a microtiter plate, or a can) from a specific through-hole of aplaten comprising a plurality of through-holes. The method includes thesteps of: a) placing the platen over the receptacle; and b) applying aburst of gas, liquid, solid, or a pin (e.g., a piston, a tube, a post, aplug) to the specific through-hole to transfer the reagent or probe intothe receptacle. The burst of gas, liquid, or solid can be generated, forexample, with a syringe, or by depositing a photodynamic or photothermalmaterial (carbon black, plastic explosives, water droplets) in or abovethe through-hole, and then exposing the photodynamic or photothermalmaterial to a laser beam of frequency and intensity suitable to activatethe photodynamic or photothermal material.

In another embodiment, the invention features a device for filling ordraining through-holes in a platen having a plurality of through-holes.The device includes: a) a holder adapted to accept the platen; b) anozzle having an aperture of a suitable size to inject a sample into asingle through-hole in said platen; and c) a valve that controls a flowof a sample through said nozzle, wherein the holder and nozzle can movewith respect to each other. The nozzle can be, for example, positionedso as to contact the platen (or not). The device can optionally includea microplate (e.g., a microtiter plate) positioned to receive samplesfrom the platen, as well as a computer that can control the valve andcontrol the positions of the holder and nozzle (and, optionally, themicroplate) relative to one other. The optional microplate, the holder,and the nozzle can, in some cases, be moved independently of each otherin at least two dimensions. Alternatively, the nozzle can be held in asingle position while the holder and nozzle can be moved independentlyof each other in at least two dimensions.

In another embodiment, the invention features a method of analyzing thekinetics of one or more reactions occurring in at least one of thethrough holes of a platen. The method includes: a) providing a firstplaten having a plurality of through-holes, wherein the through-holesare loaded with a first sample or set of samples; b) introducing theplaten into a detection device; c) introducing a second platen having aplurality of through-holes into the detection device, wherein thethrough holes are loaded with sample or reagent; d) registering andcontacting the platens such that contents of the through-holes of saidfirst platen can mix with contents of corresponding through-holes ofsaid second platen; and e) detecting a change in a physical property ofthe contents of at least some of the through-holes over time.

In another embodiment, the invention features a method of analyzing aphysical property of a sample in an array. The method includes the stepsof: a) providing a platen having a plurality of through-holes, where thethrough-holes are loaded with a sample; b) placing the platen betweentwo partially transmitting mirrors; c) illuminating the samples throughone of the mirrors (e.g., with a laser, atomic lamp, or other lightsource, including white light sources); and d) detecting optical outputfrom the sample. Optionally, mirrors that reflect at only one wavelengthand transmit at all others can be used, and non-linear optical effectscan also be observed. The “imaging” step can involve, for example,measuring light emanating from the array or measuring light emitted fromthe mirror opposite from the illumination source. The platen can also beplaced within a laser cavity, and an optical gain medium can bepositioned between the two mirrors.

The invention also features a method of measuring sample output from anarray. The method includes the steps of: a) providing a platen having aplurality of through-holes, wherein the through-holes are loaded withsample; b) introducing the sample into an array of capillaries; c)eluting the samples through the capillaries using pulse pressure,creating a non-continuous flow; d) spotting the eluting samples onto asurface that is moving relative to the capillaries (e.g., a web, a tape,a belt, or a film), wherein the spots are discrete and no mixing of thesamples occurs; and e) analyzing a physical property of the spots.

The invention also features a method of storing a plurality of samplesin an assay-ready, high-density format. The method includes the steps ofa) providing a platen having a plurality of through-holes; b) loadingthe through-holes with the samples (e.g., small molecules) dissolved ina mixture comprising two solvents, a first solvent having a low vaporpressure (e.g., dimethyl sulfoxide (DMSO)) and a second solvent having ahigher vapor pressure relative to the first solvent (e.g., ethanol;preferably, both solvents are inert and are able to dissolve thesample); and c) evaporating the second solvent to result in a pluralityof samples in first solvent (preferably as films on the walls of thethrough-holes). The volume of the first solvent in each solution can be,for example, less than about 25 nl (e.g., less than 10 nl, 1 nl, 250 pl,100 pl, or even less than about 25 pl; e.g., a “microdroplet”). In someembodiments, the sample dissolved in the first solvent forms a film onthe wall of a through-hole.

The invention features a method of forming a high throughput assay. Themethod includes: a) providing a platen having a plurality ofthrough-holes, wherein at least some of the through-holes contain asample dissolved in a solvent having a low vapor pressure (such as aarray of samples prepared for storage according to the above method); b)cooling the platen to a temperature sufficient to freeze the dissolvedsample, c) dipping the platen into a solution comprising a reagent,wherein the temperature of the solution is less than the freezing pointof the sample, but greater than the freezing point of the reagentsolution, d) removing the platen from the reagent solution, and e)warming the platen to a temperature greater than the freezing point ofthe sample. The reagent solution can be, for example, an aqueoussolution.

Yet another embodiment of the invention features a filtration device,having first and second platens, each having a plurality ofthrough-holes, and a semi-permeable membrane. The platens are alignedsuch that the through-holes of the first platen are substantiallyaligned with the through-holes of the second platen and the membrane issandwiched in between the two platens. Optionally, the platens can havehydrophobic surfaces. The semi-permeable membrane can be, for example, anitrocellulose membrane, or can include a layer of cells.

In yet another aspect, the invention provides a cell chip containingfirst and second platens, each having a plurality of through-holes, anda porous membrane, wherein the platens are aligned such that thethrough-holes of the first platen are substantially aligned with thethrough-holes of the second platen and the membrane is sandwiched inbetween the two platens. By “cell chip” is meant at least a platencontaining a plurality of through-holes and containing in at least onethrough-hole a cell and culture media.

In another aspect, the invention provides a cell chip containing inorder from top to bottom: (a) a coverslip in contact with a spacer; (b)a spacer that separates the covership from a first platen; (c) a firstplaten having a plurality of through-holes; (d) a gasket containing aplurality of through-holes that provides a seal between the firstplatent and the membrane; (e) a porous membrane containing aluminumoxide and having pores between 0.1 and 1 μm sandwiched between thegasket and the second platen; (f) a second platent having a plurality ofthrough holes; and (g) a solid support in contact with the secondplaten.

In yet another aspect, the invention provides a method of culturing acell on a cell chip, the method involving providing a cell chip of anyprevious aspect containing cell culture medium; contacting the porousmembrane with a cell; and incubating the cell under conditions suitablefor cell survival. In one embodiment, the conditions include contactingthe cell chip a gas permeable liquid (e.g., perfluorodecalin). In yetanother aspect, the cell chip further comprises a hydrophobic fluid(e.g., perfluorodecalin, silicone oil or mineral oil) in contact withthe cell culture medium.

In yet another aspect, the invention provides a method of constructing acell chip of any previous aspect, the method involving filling a firstplaten having a plurality of through-holes with cell culture medium;contacting the first platen with a porous membrane; and contacting themembrane with a second platen having a plurality of through-holes, suchthat the through-holes are substantially aligned, thereby constructing acell chip. In one embodiment, the cell chip further contains a solidsupport in contact with the first platen. In another embodiment, thecell chip further comprises a spacer in contact with the second platen,wherein the spacer is in contact with a cover slip. In yet anotherembodiment, the cell chip further comprises a gasket sandwiched betweenthe first platen. In still another embodiment, the gasket comprises aflexible material (e.g., a biocompatible elastomer, such as teflon,silicone, or rubber.

In yet another aspect, the invention provides a method for identifyingan agent having a desired biological activity, the method includingcontacting a cell chip of any previous aspect containing a cell with aplaten containing an agent; contacting the cell with the agent; anddetecting an alteration in the cell, thereby identifying an agent havinga desired biological activity. In one embodiment, the agent is presentin cell growth medium, or is contacted with the cell using a slotted pinor syringe, or by adding the cell to a well containing the agent. Inanother embodiment, the agent is a polypeptide, nucleic acid molecule(e.g., siRNA, microRNA, or an aptamer), or small compound. In anotherembodiment, the alteration is an alteration in gene expression,polypeptide expression, cell growth, proliferation or survival, in theintracellular localization of a cellular component, morphologicalchange, or change in motility. In yet another embodiment, the alterationis detected in an immunoassay, an enzymatic assay, highthroughput geneexpression profiling, reverse transcriptase polymerase chain reaction(RT-PCR), quantitative PCR, real time PCR, methylation, or high contentscreening (HCS) using quantitative fluorescence microscopy and automatedimage acquisition. In yet another embodiment, the high content screeningdetects alterations in protein translocation. In yet another embodiment,the cell is lysed and the proteins or nucleic acid molecules are boundon a binding surface. In yet another embodiment, the binding surface isa weak cationic exchange medium. In another embodiment, the boundproteins or nucleic acid molecules are analysed for a characteristicselected from the group consisting of sequence, molecular weight,binding characteristic, and expression level. In another embodiment, thebinding characteristic is detected in an immunoassay or by polypeptidebinding.

In various embodiments of any of the above aspects, the membranecomprises pores that are no more than half the through-hole diameter(e.g., pores of between about 0.2-250 μm) In various embodiments poresare about 0.2 μm, 0.5 μm, or 1.0 μm in diameter. In other embodiments ofany previous aspect, the membrane comprises aluminum oxide orpolycarbonate. In still other embodiments, the polycarbonate comprises 1μm pores. In still other embodiments of any previous aspect, themembrane is coated with fibronectin, laminin, collagen, or anothersubstrate that supports cell adhesion. In still other embodiments of anyprevious aspect, the platens comprise metal (e.g., gold, Tungsten orstainless steel), polystyrene, or a flexible material (e.g., silicone,rubber, teflon). In still other embodiments of any previous aspect, thechip further comprises a gasket that seals off individual wells, such asa removable gasket. In still other embodiments, the gasket contains aplurality of through-holes aligned with those of the platens. In stillother embodiments of any previous aspect, the chip further comprise ahydrophobic compound that prevents lateral diffusion, such as ahydrophobic compound that provides a watertight seal between themembrane and the platen. In still other embodiments of any previousaspect, the platen comprises a flexible biocompatible material and theother platen is a rigid platen that supports the flexible platen. Invarious embodiments, the flexible platen comprises silicone,polypropylene, or rubber. In still other embodiments of any previousaspect, the two platens are attached by raised surfaces on one platenthat fit into a recessed surface on the other platen. In still otherembodiments of any previous aspect, the total thickness of the cell chipis less than about 10 mm, 5 mm, or 1 mm. In still other embodiments ofany previous aspect, a solid support (e.g., a microscope slide) incontact with the first platen. In another embodiment, the chip furthercontaining a coverslip in contact with the second platen. In still otherembodiments, the coverslip, microscope slide, and cell chip are securedtogether. In still other embodiments of any previous aspect, the fillingof the chip with media is accomplished by placing the first platen on asolid support and centrifuging the platen and solid support. In oneexample, the first platen is contacted with a gasket and cell medium isthen overlayed on the platen.

A “spatially addressable through-hole” has a position and dimensionsthat are known to a high degree of certainty (e.g., relative to areference position on the device). The degree of certainty is sufficientthat the through-holes of two platens placed one on top of the other canalign, allowing reagents to transfer in a parallel fashion. The degreeof certainty is also sufficient such that a sample in any giventhrough-hole can be retrieved by a robotic device that knows only theposition in which that hole should be found relative to a referencepoint on the device. The term “planar array of through-holes” refers toan array of through-holes on a platen such as that described in PCTapplication WO99/34920.

A “reagent” is a chemical compound, a gas, a liquid, a solid, a powder,a solution, a gel, a bead, or electromagnetic radiation.

The term “probe” or “chemical probe” refers to a chemical, biological,mechanical, or electronic structure that detects a specific analyte by aspecific binding or catalytic event. The binding or catalytic event canbe transduced into a signal readable by an operator. One type ofchemical probe is an affinity probe (e.g., a specific nucleic acid thatbinds to another nucleic acid). Examples of mechanical probes include acantilever that has a ligand immobilized on its surface and a materialwhose properties (e.g., strain, inertia, surface tension) change inresponse to a chemical or biological event.

The term “chemical detection event” refers to a chemical reactionbetween molecule(s) of interest and probe molecule(s) that in turnproduces a signal that can be observed by an operator. For example, thehydrolysis of fluorescein di-β-galactoside by the enzymeβ-galactosidase, to produce the fluorescent molecule fluorescein, is achemical detection event. In some cases, the chemical detection eventcan involve a series of chemical reactions triggered by an initialinteraction of analyte and probe (e.g., activation of a signaltransduction pathway in a probe cell by the binding of a ligand to asurface receptor).

The term “linker molecule” means a molecule that has a high affinity foror covalently links to the surface of a platen or bead. The linkermolecule can have a spacer segment such as a carbon chain, and can alsohave a functional group at its end to enable attachment of probemolecules covalently or with high affinity.

The term “immobilized” means substantially attached at the molecularlevel (i.e., through a covalent or non-covalent bond or interaction).

The term “photocleavable compound” refers to a compound that contains amoiety that, when exposed to light, dissociates into multipleindependent molecules.

The term “small molecule” refers to a molecule having a mass less thanabout 3000 daltons.

The term “hybridization” refers to complementary, specific binding oftwo or more molecules (e.g., nucleic acids) to one another.

“Solid phase synthesis” refers to a chemical synthesis process in whichat least one of the starting materials in the synthesis reaction isattached to a solid material such as a polymer bead, a gelatinous resin,a porous solid, or a planar surface.

The term “blotter” refers to a material capable of capturing excessliquids by absorption.

The term “bead” means a small particle, generally less than about 1 mm(e.g., less than about 100 μm) in any dimension, with the ability tohave reagents attached to its surface or stored in its interior. A beadcan be made from one or more of a variety of materials, includingorganic polymers, glass, and metals. The reagent is typically attachedto the bead by chemical reaction with a reactive functional group suchas a carboxyl, silanol, or amino group on its surface. Reagents can, forexample, be confined to the bead by covalent chemical attachment or byphysical adsorption to the bead surface. The bead shape can be nearlyspherical, irregularly shaped, or of an intermediate shape.

The term “stringency” refers to the degree to which non-specificmolecular interactions are disrupted during a washing step.

The term “electrophoretic washing” refers to the removal ofnon-specifically bound, ionic molecules from a probe by applying anelectric field.

“Specific interactions” are interactions between two molecules resultingfrom a unique three-dimensional structure of at least one of themolecules involved. For example, enzymes have specific interactions withtransition state analogues due to their evolution toward stabilizingreaction intermediates.

The term “micro-plate” refers to a collection plate used to transfer thecontents of the through-holes of an array, where no cross contaminationof the through-holes occurs in the transfer.

The term “micro-droplet” means a drop of liquid having a volume of 50 n1or less (e.g., less than about 50 nl, 25 nl, 10 nl, 5 nl, 1 nl, 500 pl,250 pl, 100 pl, 50 pl, or less).

The term “physical properties” means any measurable property of anobject or system, including electrical, magnetic, optical, thermal,mechanical, biological, nuclear, and chemical properties.

The new methods have numerous advantages. For example, the new methodsallow optimization of processes in a parallel manner. For instance,synthesis of a particular molecular species often requires tediousquantitative investigation of different synthetic methods with a viewtowards optimizing product yield. Using the new methods, processparameters can be varied on a through-hole-by-through-hole basis in thearray, and the product analyzed to determine the protocol best suitedfor high yield synthesis.

Another advantage of the invention over conventional arrays of chemicalprobes on a planar substrate is that each chemical detection event takesplace in a physically isolated container (i.e., the through-hole),allowing amplification of the signal by catalysis (e.g., releasingdetectable molecules into the solution contained in each through-hole).Such detectable molecules include, for example, fluorescent products ofa fluorogenic enzyme substrate, and chromogenic products of achromogenic substrate. Physical isolation of samples retained in thearray also prevents cross-contamination by eliminating lateralcommunication between the through-holes.

Another advantage of the invention is that each through-hole can have aprecise and known spatial location in the array. Each through-hole isthen spatially addressable, thereby facilitating the insertion andremoval of liquids from each through-hole, the analysis of the contentsof each through-hole, and the alignment of multiple arrays for highlyparallel transfer of reagents.

Another advantage of the invention is that the relative volumes of themembers of two arrays can be easily adjusted by changing the depth ofone array with respect to the other.

Still another advantage of the invention is that substances that bind tochemical probes contained in the through-hole array can easily berecovered as distinct samples for further analysis. For example, thebound contents of the well can be eluted onto a planar substrate foranalysis by matrix-assisted laser desorption and ionization (MALDI) orsurface-enhanced laser desorption and ionization (SELDI) massspectrometry, or nuclear magnetic resonance (NMR) spectroscopy.Alternatively, the contents of the through-hole can be electrosprayeddirectly from the through-hole into a mass spectrometer. The contents ofthe through-hole can also be crystallized and analyzed with x-ray orelectron diffraction techniques (e.g., to determine crystal structure).This aspect of the invention allows for sensitive detection ofunlabelled analytes.

Yet another advantage of the invention is that the samples can beintroduced or removed from the platen by electrophoresis, as thethrough-holes can allow for conduction of an electric field.

Another advantage of the invention is that samples are accessible fromboth sides of the platens. This means, for example, that samples can beremoved from the platens by applying pressure, an air or gas stream, oran explosive charge to a through-hole of interest and then collectingthe material from the opposing face of the platen. Alternately, samplescan be sucked out of the platen without creating a vacuum. Thus, thevolume of the samples in not limited by the current state-of-the-artmicrofluidics techniques, and a minimum quantity of fluid is lost uponthe collection of the sample. A pressure can be applied, for example, inthe form of a solid pin (acting, e.g., as a piston), or in the form of aburst of inert gas. Another implication of this advantage is that it isrelatively easy to perform electrospray ionization mass spectrometrydirectly from the platen. Simultaneous measurement of luminescence fromtwo spectrally distinct luminescent probes located in the microchannelarray can be performed in either a trans- or epi-illumination opticalconfiguration, including, for example, a light source, an opticalfilter, and a CCD camera. Optical signals can be collected from bothsides of the platen simultaneously.

The numerous samples contained in the platen can be rapidly transferredto a flat surface or membrane, facilitating processes such as SELDI massspectrometric analysis and growth of bacterial cells (e.g., cellscontained in the through-holes), to form individual colonies for storageand further analysis. Transfer from a planar material to the array canalso be accomplished, as in electroblotting from a polyacrylamide 2-Dprotein gel into the array.

Advantageously, the surface area of the liquid exposed to theenvironment is minimized by the high aspect ratio geometry, thuslimiting evaporation.

Still another advantage of the new methods is that the sample containedin a given through-hole constitutes a small thermal mass and can,therefore, reach thermal equilibrium quickly and uniformly. The fact isrelevant, for example, to synthetic methods that involve heating and/orcooling steps (e.g., replication of nucleic acids using the polymerasechain reaction, PCR).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded top corner view of a platen with top and bottommasks.

FIG. 2 is a cross-sectional view of an interlocking array system.

FIG. 3 is an illustration of a method for transferring small volumedrops into specific through-holes in an array with a pin array.

FIG. 4 is an illustration of a method for producing a mask by use of UVcurable epoxy.

FIG. 5 is an illustration of a method for producing a mask by use of anarray of pins having a precision fit into a matching array ofthrough-holes.

FIG. 6 is an illustration of a method for storing liquid from individualwells from a microtiter plate in a bundle of capillary tubing fortransference into high-density through-hole arrays.

FIG. 7 is an illustration of a method for transferring fluid from wellsin a microtiter plate to through-holes in an array using a flexiblemember.

FIG. 8 is a drawing of an array in which the through-hole cross-sectionsare shaped to hold only one microsphere per through-hole, and thelongitudinal cross-section is tapered such that the microsphere sits inthe hole either at or below the array surface.

FIG. 9 is an illustration of a method for transfer with a singlesampling device with a fast sequential positioning of a mechanicalplunger over the through-holes to be sampled and pushing the plungerthrough the hole to transfer the hole's contents to the well of amicrotiter plate located at a small distance below the through-holearray.

FIG. 10 is an illustration of a method for transferring materials from athrough-hole in a through-hole array into a well of a microtiter plateusing a gas jet generated by spatially localized heating with a focusedlaser beam.

FIG. 11 is an illustration of the transfer of material from athrough-hole in the array to a well in a microtiter plate with a gas jetcaused by localized ignition of an explosive charge randomly distributedin a thin sheet overlaid on one surface of the through-hole array.

FIG. 12 is an illustration of a sheet with an explosive charge patternmatching the through-hole positions in the array.

FIG. 13 is an illustration of a method to interface massively parallelHPLC separation with inherently serial analytical methods such as massspectrometry.

FIG. 14 is an illustration of a chromatographic device.

FIG. 15 is an illustration of a linear MALDI-TOF mass spectrometer.

FIG. 16 is an illustration of an array of chamfered through-holesmachined in a block of material, a syringe bank with the samecenter-to-center spacing as the through-hole array, wherein the syringeneedles pass through a metal block that is attached to the syringe bankholder by pneumatically-actuated, spring-loaded pins.

FIG. 17 is an illustration of the array of FIG. 16, wherein thecapillary channels are pressurized by the syringes.

FIG. 18 is an illustration of an array similar to that of FIG. 17, withthe exception that the syringe bank is bolted to the capillary tubearray.

FIG. 19 is an illustration of an array similar to that of FIG. 18, withthe exception that the syringe bank is bolted to the capillary tubearray.

FIG. 20 is an illustration of a method of manufacturing a platen havinga plurality of through-holes and two opposing surfaces, by bondingtogether multiple grooved surfaces.

FIG. 21 depicts an array positioned inside an optical resonatorfeaturing a source of illumination and two partially reflectivesurfaces.

FIG. 22 depicts a device for removal of the contents of a through-holearray and transfer of those contents. The device has a nozzle, a stagefor holding the through-hole array and a stage for holding a capturechamber. Movement in two dimensions of the nozzle or the through-holearray can be achieved.

FIG. 23 depicts a method for wiping excess fluids from the surface ofthe platen. The device has enables a through-hole array to be loadedwith sample and removed through a wiping fluid in an efficient manner.

FIG. 24 depicts a device for aligning platens having a plurality ofthrough-holes inside a detection device, wherein the platens are held inplace through device comprising two pins attached to a flat base.

FIG. 25 shows a flow chart of a cell-chip microarray. A platen of rigidmaterial such as metal or polystyrene with pores of 50-200 μm indiameter is attached to a porous membrane forming an array of microwellswith a porous bottom. Cells are added and allowed to adhere to themembrane before test compound is added with a microspotting pin. Afterincubation, the membrane may be processed for analysis of protein ormRNA expression, or any assay or assay component of interest.

FIG. 26 shows polycarbonate membranes, having 1.0 μm pore size, soakedin 100 ug/mLl fibronectin and air dried for 2 hours. The membrane wasattached to the bottom of a Petri dish by the addition of 10% agarose atthe edges. PKCβ-GFP transfected 293 cells were added at a concentrationof 5×10⁵/mL and incubated 37° C., 5% CO₂. Images were acquired byconfocal microscopy.

FIG. 27 shows polycarbonate membranes, 1.0 μm pore size, soaked in 100ug/mL fibronectin and air dried for 2 hours. The membrane was attachedto the bottom of a Petri dish by the addition of 10% agarose at theedges. PKCβ-GFP transfected 293 cells were added at a concentration of5×10⁵/mL and incubated 37° C., 5% CO₂. Then, 1 uMphorbol-12-myristate-13-acetate (PMA) was added. After 1 hour at 37° C.,5% CO₂, images were acquired by confocal microscopy.

FIG. 28 shows attachment of cells to the membrane in the cell-chipdevice comprised of gold platen and porous membrane assembled asdemonstrated in FIG. 1.

FIG. 29 shows microspotting on gold platen. A 300 mesh gold platen (50um² holes) was wetted with medium. Fluorescein in 10% DMSO/PBS wasspotted on platen using a FP9 floating pin (VP-scientific.) Spots wereexamined under a confocal microscope at 5× magnification.

FIG. 30 shows cell uptake of Hoechst stain. PKCβ-GFP cells wereincubated overnight in 300 mesh gold platen (50 um² holes) and washedwith medium. Hoechst stain was then spotted on the platen that was driedby blotting.

FIG. 31 shows microspotting on a gold platen. A 300 mesh gold platen (50um² holes) was wetted with medium or briefly dried by blotting. A dropof mineral oil was added to the platen before adding Fluorescein in 10%DMSO/PBS using a FP9 floating pin. Spots were examined under a confocalmicroscope at 5× magnification.

FIG. 32 shows cell uptake of Hoechst stain by PKCβ-GFP cells that wereincubated overnight in a stainless steel cell-chip and then washed withmedium. Hoechst was spotted on a first platen that was dried byblotting. The stainless steel platen (National Jet Company, LaVale,M.D.) tested was a 1-inch square and 400-μm thick with pores 150 μm indiameter. A second platen was attached using adhesive sealing film withthe center cut out. The chip was placed in a cytospin sample chamberwith the funnel removed with a small gasket between the platen and thetop of the chamber.

FIG. 33 shows an anopore membrane sandwiched between two stainless steelplatens (as shown in FIG. 8). PKCβ-GFP cells were added and incubatedovernight. Note unequal distribution of cells in the well, possibly dueto leaks of media between platen and membrane

FIG. 34 shows a cell microarray prototype based on a 200-μm thickTungsten platen. The platens (National Jet Company, LaVale, M.D.) hadpores of 300 μm in diameter and were attached with 4 screws.

FIG. 35 shows cell culture on an anopore membrane sandwiched between twotungsten platens (as shown in FIG. 34). PKCβ-GFP cells were added andincubated 48 hrs. Note more random cell distribution with more secureattachment; however, the platen is not yet optimized for cellattachment.

FIG. 36 shows an open array-based cell chip and delivery ofC12-resazurin to a single well on the array using a floating pin.

FIG. 37 shows an open array-based cell chip with PKCβ-GFP transfectedcells, added at 5×10⁵/mL and incubated 37° C., 5% CO₂. Images wereacquired by confocal microscopy.

FIG. 38 shows essentially that which is depicted in FIG. 12, except herethe platen is not shown.

FIG. 39 shows one particular embodiment of the invention—specifically,an apparatus for in vitro and ex-vivo analysis. A platen of rigidmaterial such as metal or polystyrene with pores of 10-300 μm indiameter is attached to a porous membrane or modified glass surfaceforming an array of microwells with a porous bottom. Cells are added andallowed to adhere to the membrane overnight before test compound isadded with a micro spotting pin. After incubation, high content imageanalysis is performed. Membranes are processed for analysis of proteinor mRNA expression, or other assay or assay component of interest.

FIG. 40 is essentially that which is depicted in FIG. 15, but here showsthe cross-section of an individual well.

FIG. 41 shows structural enhancements to increase pressure on the memberand/or gaskets to seal off individual wells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods of creating, storing, and screeningdiverse chemical and biological compositions, each contained in athrough-hole that traverses a platen, as well as methods for making andusing platens, particularly platens containing cells. In certainembodiments, the methods include transmitting reagents to a selectedgroup of holes in a dense array of through-holes. Additional rounds ofreagent transmission are provided as needed. The invention also providesfor placing a series of masks over a planar array of through-holes andflowing reagents through the masks to build a defined pattern of probesor reagents such that the contents of each through-hole can be known. Inan alternate embodiment, the invention provides distributingprobe-holding particles, such as beads or cells, into the array ofthrough-holes. Such probes include, but are not limited to, nucleicacids, peptides, small molecules, and chemical sensing cells. Uses ofthe arrays include screening of genetic libraries, producing andscreening compound libraries for discovery of pharmaceutical leads,optimization of reaction conditions, gene expression analysis, clinicaldiagnostics, genomics, functional genomics, pharmacogenomics, structuralgenomics, proteomics, production and optimization of industrialcatalysts, chemical genetics, identification of suitable conditions forreactions (e.g., conditions suitable for protein crystallization),genotyping, polymorphism analysis, examination of RNA expressionprofiles in cells or tissues, sequencing by hybridization, andrecombinant enzyme discovery.

A platen having a high-density array of through-holes in accordance withone embodiment of the present invention is illustrated in FIGS. 1 (topview) and 2 (cross-sectional side view). The platen can be made ofsilicon or other rigid materials, such as metal, glass, or plastic. Theplaten material can be chemically inert, or can be rendered so byappropriate surface treatments.

Referring to FIG. 1, each through-hole has a square cross-section,although circular or rectangular cross-sections can alternatively beused. The diameter of each through-hole is less than 1 mm (e.g., lessthan about 600 μm, 300 μm, 100 μm, 10 μm, 1 μm, or 100 nm), typically200-250 μm, and the depth of the platen can be 10-2000 μm or more,generally about 250-1000 μm.

Greater depths can be achieved using a bundle of glass capillaries.Alternatively, platens having greater depths can achieved by bondingtogether multiple surfaces having parallel grooves, creating a longthree dimensional object having through-holes running throughout thelength of the object. This object can be subsequently slicedhorizontally, allowing flexibility in the depth of the through-holes.The result is the ability to use arrays with compatible positions ofthrough-holes, wherein the depths of the through-holes can vary fromarray to array.

For spatial addressability, center-to-center spacing of through-holesshould be fairly precise. Hole-to-hole spacing depends on the dimensionof the through-holes within the platen. The though-holes can be arrangedin regular rows and columns, hexagonal arrays, or other configurations(e.g., groupings of through-holes into smaller sub-arrays). Multipleplatens can be fabricated with the same arrangement of through-holes sothat the pattern is reproducible, and each through-hole can beidentified by its own address within the array.

When three platens having through-holes are stacked, the total volume ofa single channel (i.e., three through-holes stacked) is typically ˜100nl. Using this volume as an example, if the entire channel were filledfrom a dense yeast cell culture (˜10⁷/ml), each channel would thuscontain approximately 10³ yeast cells. Based on a yeast cell volume of70 μm³, the maximum number of cells per 100 nl channel is on the orderof 10⁶. A minimum of 100 cells per microchannel can be adequate tocompensate for cell-to-cell variability of yeast cell response to thebioassays. However, this volume can vary depending not only on thediameter of the through-holes, but on the depth of the through-holes.This ability to vary the volume of samples allows flexibility, enablingthe use of a wider variety of materials, concentrations, and reactionconditions.

Optional features such as binary identification codes, or holes andgrooves for indexing and alignment, can also be incorporated into eachplaten.

I. Methods of Making Devices Having Arrays of Through-Holes.

Fabrication of an Array of Through-Holes by Casting in Resin.

Conventional technologies for manufacturing high-density through-holearrays include micro-machining, electrospark discharge machining (EDM),or chemical etching. Alternatively, the arrays can be cast in a polymeror resin. A casting mold can be designed such that the inner diameter ofthe mold will be equal to or larger than the final outer diameter of thearray device. The depth of the casting mold can be as little as 0.5 mmfor a single array, or 1 meter or longer. In the case where a long blockof resin is cast, the resin can be cross-sectioned into slices ofdesired thickness and the surfaces can be polished or smoothed. Thethrough-holes can be defined in the cast by several methods. Solid wire,fiber, or an array of pins of the desired geometry and diameter can bearranged within the casting mold. If necessary, the wire, fiber or pinscan be immobilized in place with the use of one or more positioning jigswithin the casting mold. The chemistries of the wire, fiber, or pinsmust be chosen such that they will not form a permanent bond with theresin or polymer as it solidifies, so that they can be pulled out toproduce the through-holes. For example, the fibers may beethyleneterephthalate and the resin is polymethylmethacrylate (PMMA).Alternatively, the surfaces of the wire, fiber, or pins can be coatedwith a release agent such as an oil, a fluoropolymer, water, or apolymerization inhibitor that will facilitate the removal of the wire,fiber, or pins from the cast resin or polymer once the final curing,setting, or polymerization is complete.

In an alternate system, the through-holes can be defined by positioningan array of hollow tubing or capillaries within the casing mold. Thehollow tubes can be immobilized within the casting mold in a positioningjig. Use of tubing of different internal diameter results in an arraywith through-holes of different diameters. The chemistry of the hollowtubes and polymer will ideally be chosen such that a permanent bond willform between the outside hollow tube and the resin or polymer that iscast. The inner surface of the hollow tubes will then make up thethrough-holes of the array. The hollow tubes can be made of glass orfused silica, a polymer, or a metal.

The chemistry of the resin or polymer that is cast can be selected suchthat the surface of the array device is of a desired hydrophobic orhydrophilic character. The chemistry of casting resins, such as acrylateor polystyrene, can be modified with hydrophobic groups to result in anarray with the desired surface chemistry. Alternatively, the surfacechemistry of the array device can be modified with standard techniquesafter slicing and polishing. In addition, the chemical or physicalproperties of the polymer can be modified by the addition of othermaterials. For example, to control the electrical conductivity of thedevice, particles of a conductive metal can be mixed into the resin orpolymer prior to casting the mold to confer conductivity to the device.Generally, the more metal particles that are mixed with the resin orpolymer prior to casting, the greater the conductivity of the deviceswill be.

Additives to the resin or polymer can be used to improve the sensitivityof optical imaging of the array. For example, metal particles can beadded to make the material between the through-holes. The metalparticles enable light to scatter, causing a fluorescent signalgenerated by a probe in a through-hole to reflect toward the detectorand to prevent cross-talk of signals. Alternatively, carbon black may beadded to make the material, preventing cross-talk and minimizing signalfrom light scattered off the surface of the array. More preferably, acombination of a light scattering agent such as titanium dioxide and alight absorbing agent, such as carbon black are added to the resin orpolymer to achieve maximum optical density between the holes.

Using hollow tubes in the casting mold to form through-holes allows thechemical properties of the tubes to be varied according to the needs ofthe application. Tubes manufactured from a biologically inert polymer(e.g., polyetheretherketone (PEEK) or poly(tetrafluoroethylene) (PTFE))are desirable for some applications. Alternatively, fused silica tubingcan be used to form the through-holes. The interior surface of the fusedsilica can be derivatized prior to casting, allowing for virtually anylevel of desired hydrophilicity or hydrophobicity. A metal or alloytubing can also be used to form the through-holes of the array. Metaltubing can be coated to make it biocompatible. For example, metals canbe coated with thin layers of gold and the gold surfaces can be readilycoated with a variety of reagents possessing thiol moieties.

The inner surfaces of the tubes or capillaries can, for example, becoated with materials that facilitate the use of the resulting slices asa probe array. For instance, each tube or capillary can be coated with adifferent nucleic acid probe, so that when the block of resin is sliced,the resulting platens can be used as genosensors. Alternatively, theprobes can be immobilized on a porous material contained in thecapillaries.

Once a block is cast from the desired resin or polymer, it can be slicedto an appropriate thickness to form a platen having an array ofthrough-holes. In certain embodiments, the thickness of the platenranges from 0.2 to 25.0 mm. However, using this technique, arrays ofthrough-holes can be manufactured having the same length as the castingmold that is used. If desired, casts that are many meters in length canbe prepared. Standard techniques for producing silicon wafers by slicingand polishing silicon ingots in the semiconductor industry can bedirectly applied to the manufacture of an array of through-holes.Flowing a coolant through the through-holes in the cast array whileslicing can prevent heat buildup that could otherwise melt the polymeror degrade coatings or probes inside the holes. Examples of coolantsinclude cold water, cold aqueous ethylene glycol, and cold isopropanol.

If solid wires are arranged in the cast and the resulting block is thensliced, the wires can be eroded by electrodeposition onto a plate in anelectrochemical cell or by chemical degradation such as by placing theslice in concentrated nitric acid to give an array of through-holes. Theslices can be bonded to a metal sheet, the polymer eroded, and theslices used as an electrode for production of through-hole arrays bysink-EDM. The polymer can be eroded by chemical means, melted, or burnedoff.

Method of Making an Array by Stacking and Bonding Multiple GroovedSurfaces.

One method of manufacturing an array of through-holes is to stack andbond together grooved plates having upper and lower surfaces, creating athree-dimensional array. Any number of materials can be used tomanufacture the array of through-holes including, but not limited to,silicon, glass, plastics, resins, or metals. Depending on the materialused, grooves can be machined into the individual surfaces using avariety of techniques (for example, micro-machining, chemical etching,embossing, or stamping). The depth and width of each groove determinesthe dimensions of the through-holes in the completed platens and can bemachined according to the desired specifications.

A precise layering or stacking of the grooved plates into athree-dimensional array can be accomplished with the use of an externalor internal jig into which each surface is precisely placed.Alternatively, registration devices, (for example notches, posts,tongues, etc.) can be precisely integrated into each surface tofacilitate accurate stacking. After stacking the individual groovedsurfaces, they are bonded together in a permanent manner. The use oftraditional adhesives to bond the plates together is a disfavoredapproach because excess adhesive can migrate into the grooves and resultin the blockage of some of the through-holes. A preferred method for thebonding process is the use of a combination of elevated temperature andpressure, resulting in a fusion of the chosen materials. In some cases,one or both surfaces of the grooved platens are coated with a material(for example, gold) that, upon the application of an appropriate amountof temperature and pressure, diffuses into the surfaces and results in apermanent bond. The grooved plates can be made from materials thatinclude a thermoplastic, a ceramic, a glass or a metal such as silicon.

In a preferred embodiment, the width of each of the grooved plates isequivalent to the width of the final platen of through-holes, but thelength of each grooved plate is much larger than the desired thicknessof the final platen of through-holes. This results in athree-dimensional array of through-holes that is much thicker thanrequired. Individual platens of through-holes can then be precisely cutfrom this thick block to the desired specification. The platens canfurther require polishing after they have been cut in order to yield anoptically flat surface. The required surface chemistries are then beapplied to the platens. An advantage of this method of manufacturing isthe creation of platens with straight-walled through-holes that are muchdeeper than those made using traditional micro-machining technology.

Minor misalignments in the stacking of the individual grooved plates canresult in small imperfections in the registration of the through-holesin the final platens. This can interfere with operations using theplatens of through-holes (for example, stacking of two or more platensto initiate massively parallel reaction). However, even if minor errorsin positional registration exist, adjacent slices cut from each thickarray of through-holes will be a near-perfect match and the requiredstacking operations can be accomplished.

Formation of a Silicon Oxide Layer.

In one embodiment, a dense array of through-holes is produced in asilicon wafer. A silicon oxide layer is created uniformly on allsurfaces of the array by heating the silicon array in a furnace to atemperature high enough to cause oxidation. Oxygen and/or humiditylevels in the furnace can be raised above the ambient to speed oxidationprocess (see, e.g., Atalla et al., The Bell System Technical Journal,pp. 749-783, May 1959). The silicon oxide layer is advantageous becauseit enables the application of various chemical surface treatments to thearray surfaces. Examples of surface treatments are found in ImmobilizedAffinity Ligand Techniques (Hermanson et al, Academic Press, San Diego,1992) and technical literature available from United ChemicalTechnologies, Inc., Bristol, PA. Use of silicon oxide provides andadditional advantage, allowing the optical reflectivity of the surfacesto be controlled by adjusting the thickness of the silicon oxide film(see, e.g., Principles of Optics, M. Born & E. Wolf, Pergamon Press,1980, pages 59-66).

II. Methods of Modifying the Surfaces of Array Devices and Walls ofThrough-Holes.

The surfaces of the through-hole arrays can be modified in various ways(e.g., to change their physical and chemical properties). Types ofsurface modification can include, but are not limited to, theapplication of polymer coatings, deposition of metals, chemicalderivitization, and mechanical polishing.

Selectively Modifying the Surface Chemistry of Array Faces:

In order to prevent aqueous solutions from adhering to array surfacesand cross-communication between the various through-holes during loadingand other manipulations, it is desirable to coat the surfaces of theplaten with a hydrophobic coating. It is also desirable to coat theinner surfaces of the through-holes with a hydrophilic coating so thatthey retain fluids. The inner coating can further be blocked, preventingnon-specific binding, or derivatized with affinity ligands. Thiscombination of hydrophobic surfaces and hydrophilic through-holesprevents aqueous solutions from adhering to the surfaces of the arraywhile allowing instantaneous loading of the through-holes.

Generally, a platen having a dense array of through-holes is produced insilicon and coated in silicon oxide by oxidation. The platen is thencleaned, removing organic materials, by soaking in a mixture of hydrogenperoxide and sulfuric acid, or other caustic solvent cleaning solution.This treatment results in clean silicon oxide with a high surfaceenergy. Hydrophobic coatings produced using this method are stable underhigh humidity and they can be used repeatedly.

The top and bottom faces of the arrays are made hydrophobic throughexposure to vapor from a solution containing an appropriate silanizingagent (such as polydimethylsiloxane sold as Glassclad 216 by UnitedChemical Technologies, Inc.) in a volatile solvent. The silanizing agentreacts with the hydroxyl and silanol groups on the array surface andcreates covalent bonds to hydrophobic alkyl groups. Selectivemodification of the surface chemistry can be achieved by selection ofalternative silanizing agents.

In one surface modification method, a positive pressure of inert gas isapplied to the surface of the platen opposite the surface being treated.The positive pressure within the through-holes prevents the silanizingvapor from reaching the interior through-holes. Silanizing agentssuitable for rendering glassy surfaces hydrophobic includealkyltrichlorosilanes and alkyltrimethoxysilanes, many of which arecommercially available.

In another method, a first platen is made from silicon, and thenoxidized. A second silicon platen is produced with identical size andalignment holes, but without through-holes. The through-hole arrayplaten is placed on top of the solid platen on the stacking jig suchthat each through-hole becomes a well. The jig and plates are thentreated with a surface-modifying chemical reagent. The air trapped atthe bottom of the wells prevents the fluid from entering the wells.After sufficient time for the reaction to occur, the plate is removedfrom solution, dried, and heat-treated if appropriate. The backing isremoved, the array is flipped over, and the process repeated, coatingthe second surface.

Yet another method for creating hydrophobic exterior surfaces is toplace the array on a matching array of pins such that each single pinnarrowly passes through its corresponding through-hole. This array ofpins can be the electrode used to create the array of through-holesusing sink EDM. Next a coating of a hydrophobic polymer such aspolypropylene or Teflon is deposited on the exposed surfaces using gasphase deposition method such as evaporative vapor deposition. The arrayof through-holes is removed from the matching pin array, inverted, andthe coating process is repeated to cover the opposite surface.

Another method for producing hydrophobic coatings on the platen surfacesinvolves coating platen surfaces with a metal such as gold (thenexposing the arrays to a chemical that selectively reacts with themetal, but not with the uncoated through-hole surfaces. For exampleelectron beam vapor deposition can be used to coat the outer surfaces ofa platen containing a plurality of through-holes with gold, other metalor semi-conductor. Electron beam vapor deposition will preferentiallydeposit the gold on surfaces normal to the beam direction. Thegold-coated surface can then react with alkane thiols to attach ahydrophobic alkyl groups (Z. Hou et al., Langmuir, 14:3287-3297, 1998).The inner surfaces of the through-holes that are not coated with goldwill remain hydrophilic. Alkane thiols are also reactive towards othermaterials including silver, copper and gallium arsenide (Y Xia and G. M.Whitesides, Annu. Rev. Mater. Sci., 28: 153-84, 1998). Amphiphilesbesides alkane thiols can be chemically reacted to other inorganic solidmaterials to produce hydrophobic coatings. Examples of such coatingsinclude, but are not limited to, alkanephosphates on metal oxides (D.Brovelli et al., Langmuir, 15:4324-4327, 1999 and R. Hofer et al.,Langmuir, 17: 4014-4020, 2001), and alkane carboxylates on alumina (P.E. Laibinis et al., Science, 245: 845, 1989).

Selectively Modifying the Surface Chemistry of Through-Hole Surfaces.

In one method, multiple, identical through-hole arrays are prepared,aligned, and stacked. A chemical reagent is passed through thecontinuous channels formed by the stacked arrays. The reagent can be asolution, a suspension, a liquid, a vapor, or fine powder. The stack ofarrays is then washed, dried, and heated, as appropriate for theparticular coating. The stack of chips is then physically separated fromone another and the arrays on the top and bottom of the stack(“sacrificial arrays”) are discarded.

Another method for selectively coating through-hole surfaces involvesusing a robot to position a fine needle or an array of fine needlesproximal to the entrance of each through-hole. Chemicalsurface-modifying reagents can then be delivered through thisneedle/capillary directly into individual holes

In another method, all surfaces of a silicon through-hole array arechemically modified. The array faces are then mechanically polished torestore the original surface character. The array faces can then becoated again.

In still another method, the array faces are coated with a material thatis inert towards the desired surface-modifying chemicals, and then theentire array is exposed to it. For example, a gold coating can beapplied to both faces of an oxidized silicon through-hole array byelectron beam deposition. The array can then be submerged in a solutionof chemical reagent such as a silanizing reagent that reacts selectivelywith the siliceous through-hole surfaces.

In another method, the inner and outer surfaces of a through-hole arrayare coated by filling the holes with a removable, impermeable material.For example, the interior surfaces of the through-holes in an array canbe protected by filling them with a solid that can later be removed.Examples of such a solid include a wax, a plastic, a frozen oil, ice,dry ice, or a polymer such as poly(ethylene-glycol). In one example, thethrough-holes are filled, excess material removed from the surface ofthe platen, and the surface coated, leaving the interiors uncoated.Methods for removing excess materials include scraping, sanding,polishing, dissolving with a solvent, melting or burning. The solidmaterial in the interior of the through-holes can be removed undersimilar conditions. The interiors of the through-holes can then beselectively coated or modified.

Derivatizing Through-Hole Surfaces.

In many cases it is desirable to immobilize probes on the inner walls ofall, some, or one of the through-holes. There are many techniques forcovalently attaching probes to glass or plastic surfaces (see, e.g.,Immobilized Affinity Ligand Techniques, Hermanson et al., AcademicPress, 1992). Those methods useful for glass can also be used for theoxidized surfaces of a silicon substrate. For example, the inner wallsof the holes in an oxidized silicon through-hole array can be reactedwith g-glycidoxypropyl trimethoxysilane in the presence of acid andheated to provide a glycerol coating. This glycerol coating can then becovalently linked to peptide or nucleic acid probes.

Rendering the Inner Walls Porous:

The inner wall of a through-hole can be made porous by chemical etchingfollowing the procedure as outlined by Wei et al. (Nature, 399:243-246,1999). The larger area of the porous region increases surface area andthus the amount of chemical reagent attached to the through-hole innerwall. When used for synthetic transformation, the increased reagentloading of porous through-holes increases yield. When used fordetection, the increased reagent loading increases sensitivity.Furthermore, the material between adjacent through-holes can be madeporous allowing for communication between through-holes by liquids orgases. This method can be useful for the controlled delivery of reagentsstored in adjacent through-holes, allowing mixing of reagents andreactions to occur. All or part (e.g., just the middle portion) of thethrough-hole can be made porous.

Polymer Scaffolding for Protein and Cell Immobilization in a Platen

The interior walls of the through-holes of the platen can be derivatizedto allow covalent or non-covalent attachment of proteins or cells. Anysignal arising from the protein or cell thus attached is then confinedto the perimeter of the well, unless the signal is enzymaticallyamplified, as in an ELISA assay. Even if amplified in this way, however,the signal may be weakened, for example, by low analyte concentration.In the case of cell attachment, because the wells are bottomless, cellscan attach only to the interior walls and can grow upwards in twodimensions. An alternative to passive protein adsorption and covalentattachment to the walls of the array through-holes is the introductionof a three-dimensional hydrophilic scaffold such as a hydrophiliclinear, gel or foam polymer filling activated for protein coupling orcapable of protein or cell entrapment within the well.

Covalent Attachment of Proteins to Polymer-Filled Through-Holes of thePlaten

Because the interior surface of the through-holes is hydrophilic, ahydrophilic or water soluble pre-polymer can easily be loaded into thewells of the platen and the polymerization reaction can be initiated bya change in temperature or pH or by the addition of initiator. Proteincoupling can be carried out during polymerization, provided that thepolymerization conditions do not affect protein structure and function.Alternatively, protein coupling can be carried out after polymerization.Proteins can be coupled using any of a variety of reactions, includingreactions of free amines, free carboxylic acid groups, and free sulfidegroups. Reactions that form isourea linkages, diazo linkages, or peptidebonds are among those typically used to couple proteins to surfaces, butany aqueous based polymer reaction that is easily controlled can beused. Examples of polymers scaffolds include dextran and polyamides.

Dextran is a polysaccharide polymer that is very hydrophilic. The sugarresidues of dextran contain hydroxyl groups, which can be chemicallyactivated for covalent bond formation. Hydroxyl groups also formhydrogen bonds with water molecules, and thereby create an aqueousenvironment in the support. When activated with an aldehyde, dextran canbe easily coupled with proteins via amine groups (e.g., using sodiumcyanoborohydride). When activated with hydrazide, dextran can be coupledwith proteins via aldehyde or carboxyl groups using1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride) (EDC).However, use of dextran for certain applications is limited by itssusceptibility to microbial attack.

An alternative method for covalent immobilization of proteins or enzymesis through various derivatized polyamides, such as Nylon®. Polyamidesare best suited for the immobilization of high molecular weightsubstrates. Chemically, many polyamides are thermoplastic polymers withhigh mechanical strength, superficial hardness, and resistance toabrasive conditions caused by the intermolecular hydrogen bondinteractions established between the amide groups of parallel chains.These characteristics make polyamides useful for immobilized enzymes,because they provide a favorable hydrophilic microenvironment to supportboth catalytic activity and enzyme structure. Proteins covalentlyattached to a polymer scaffold are amenable to conventional biochemicalassays such as ELISAs, binding assays, and activity assays.

Encapsulation of Proteins (or Cells).

Both proteins and cells can be immobilized by encapsulation within aweb, matrix, or pores of semipermeable membranes, gels, or foams.Encapsulation of cells requires special consideration of the followingfactors: diffusion of materials within and through the support;non-toxicity of the starting materials and of the polymer to cells; andphysical properties such as optical clarity, temperature stability,flexibility, and resistance to chemical and microbial attack. Thesupport is preferably resilient and flexible, capable of hydrogenbonding, and resistant to proteolysis and hydrolysis.

Given an appropriate scaffold, mammalian cells can be cultured in threedimensions in a platen through-hole, enabling both the performance ofmany types of cell-based assays and the potential for imaging of cellswithin the through-hole using confocal techniques. Examples of types ofassays that can be carried out include reporter gene assays, cell growthassays, apoptosis assays, and assays involving events occurring at thecell surface or within the cytoplasmic region (e.g., measurements ofcalcium efflux from the endoplasmic reticulum). Such assays are usefulfor functional studies of chemical and biological libraries. Examples ofmaterials amenable to encapsulation include Poly-(2-hydroxyethylmethacrylate) (poly-HEMA) gel, Poly(carbamoyl sulfonate) (PCS)hydrogels, and Phosphorylated polyvinyl alcohol (PVA) gel.

Poly-(2-hydroxyethyl methacrylate), “poly-HEMA,” gel has superiormechanical properties, temperature tolerance, and resistance tomicrobial attack than many natural polymers (e.g., gelatin, agarose,carrageenan, cellulose, and albumin). Poly-HEMA gels can be used forentrapment of both proteins and cells. The process of biocatalystimmobilization in poly-HEMA hydrogels generally allows for highretention of immobilized enzyme activity, well-controlled porosity(e.g., to ensure sufficient mass transfer of reactants and products),and good chemical resistance. Poly-HEMA hydrogels also protect entrappedproteins and cells from bacterial degradation, by resisting bacteriaentry into the gel support. Additionally, Poly-HEMA hydrogels can retaina large quantity of water, providing a microenvironment thatapproximates in vivo conditions.

Poly(carbamoyl sulfonate), “(PCS) hydrogels,” afford adjustable gelationtime, high mechanical stability, and resistance to microbial attack. PCShydrogels also have a high degree of flexibility, which can be exploitedfor filtering or expelling samples.

Phosphorylated polyvinyl alcohol, “phosphorylated PVA” gels can be usedto immobilize cells of bacteria or yeast. These gels are economical,nontoxic, durable and support a high cell viability. Applications usingphophorylated PVA gels are limited by their poor gas permeability.

Growth of Cells on Membranes

The invention further methods for growing and analyzing eukaryotic cellsin a format where a porous membrane is sandwiched between platens whoseholes are aligned on both sides of the membrane. Cells are grown in thewells formed by one of the platens holes and the membrane. A removablegasket may be placed on top of the device to allow cells to be added.Test compounds may be introduced to the wells on either side of themembrane with micro-spotting pins or by aligning a solid surfacepre-spotted with test compounds over the wells. The device may becovered on both sides with cover slips separated by spacers to reduceevaporation. A solid substrate, such as a glass microscope slide may beused to support the device. Image analysis can be performedmacroscopically with conventional scanning devices or microscopicallyusing light or fluorescence detection. Biochemical analysis may be doneon supernatants or cell lysates using standard centrifugal or vacuumfiltration devices.

In particular embodiments, platens used to create micro-wells aresufficiently rigid to make an even contact on the membrane. The materialis preferably biocompatible and may be metal, plastic or ceramic. Theholes formed by the platen may be less than 0.5 mm in diameter spacedless than 0.1 mm apart, or any other configuration and dimension thatallows growth of adherent eukaryotic cells. To prevent lateraldiffusion, a watertight seal may be formed between the membrane and theplaten. This may be accomplished by coating the platen where it contactsthe membrane with a hydrophobic substance or by using a secondary platenmade of a flexible biocompatible material such as silicon, rubber, orother suitable material supported by a rigid platen on top. The twoplatens may be attached by raised surfaces on one platen fitting intorecessed surfaces on the opposing platen. The total platen thickness oneither side of the membrane whether a single rigid platen or a rigidplaten on a flexible platen is typically less than 1 mm.

In related embodiments, adherent cell growth is maintained oncommercially available membranes or any biocompatible porous membranethat will allow cell attachment. Optimal cell growth for adherent cellsis maintained on membranes exposed to nutrient containing medium oneither side.

The construction of the chip with medium and cells begins by filling thethrough-holes in a first platen with medium. This may be done by placingthe first platen on a solid support. A gasket may be placed on theplaten and cell medium is then overlayed on the platen. Aftercentrifugation, the air in the platen holes is displaced with medium.Next, a pre-wetted porous membrane is place on top of the first platenfollowed by placement of a second platen(s). Guide holes in all of theplatens will allow holes to be aligned visually. Alternatively, pinsattached to the bottom support spaced in the same configuration as theguide holes on the platens may be used to line up the platens. Afterplaten assembly, a gasket may be placed on top of the platen and mediumcontaining cells id added. After centrifugation to allow cells to enterthrough-holes, excess medium may be removed. Spacers (˜<0.5 mm thick)may be placed on the edges of the top platen followed by a cover slip.The device is then clamped together and placed in a humidified chambercompatible with cell growth.

Pre-spotted test compounds such as proteins, small molecular weightcompounds, RNAi or other nucleic acids may be applied to the cells usinga solid substrate with compounds arranged in the same configuration asthe platen through-holes. In such embodiments, the compounds are spottedon a solid substrate in a medium with low volatility such as glycerol toreduce evaporation. The compounds are then applied to the solidsubstrate to have spatial orientation as the platen through-holes. Thesolid substrate to which the compounds are applied may also have a guidepin that fits into the cell-chip so that each hole is aligned with acompound. In addition, a layer of mineral oil may be applied to eitheror both platens prior to compound contact to prevent lateral diffusionand cross contamination of compounds. Contact of the compound into thethrough-holes of the platen then allows diffusion into the through-holesand dispersal to cells attached to the membrane. Cells may then beincubated until analysis.

In other embodiments, analysis after addition of test compound mayinclude image and/or biochemical analysis. Image analysis may be donewith existing technologies such as visible light, laser scanners,visible or fluorescence microscopy to detect morphological changes inthe cell or biochemical changes by visible or fluorescent dyes.Biochemical components of the cell lysates or supernatants such as RNAor protein may be analyzed by existing technologies such as centrifugalor vacuum filtration to separate components onto a membrane or modulefor analysis by qPCR, or SELDI MS.

In other embodiments, compounds and other test substances are deliveredusing a standard microarray-spotting pin. The micro format allows highthroughput screening using an array spotter. Cell viability ormorphological changes can be measured directly on the membrane. Proteinor mRNA expression can be determined after membrane processing.Applications of this technology include lead generation screening fordrug discovery or gene function studies using RNAi, cDNA and RNA.

Fiberglass Chip

A through-hole array having holes filled with fibers can be created in asimple and inexpensive manner. Examples of useful fibers includefiberglass filters, mesh glass fiber filters, and polymer filters suchas Nylon® and polyethersulfone. These materials can be surface modifiedfor specific interactions prior to fusing.

In one method, a sheet of loose fiberglass mesh is fused betweenthrough-holes of an array. The fusion can be achieved by pressing thefiberglass against a through-hole array created from silicon or othersuitable metal that has been heated to a temperature sufficient to fusethe fiberglass, and then removing the array. Non-reactive lubricantssuch as graphite or molybdenum grease can be used to aid in theseparation of the heated array and fiberglass arrays. Alternately, thefiberglass sheet can be pressed between two, aligned, heatedthrough-hole arrays.

The resulting fiberglass array can be treated to make the regionsbetween the porous holes hydrophobic. Such treatment can be accomplishedby blowing gas through the array while exposing the opposing face to asilanizing vapor. The porous areas in the resulting fiberglass array canbe used for immobilizing probes such as nucleic acids or peptides withthe advantage of very high surface areas for attachment, ease of flowthrough the array and the optical transparency of glass.

An alternate method for producing the fiberglass arrays is to inject orcause a polymerization of a thermoplastic material in the space betweenthe desired holes, for example, by using a printing technique to deposita polymer, epoxy, monomer, or polymerization initiator, or byphoto-initiating polymerization or curing in the desired areas using oneor more photomasks.

Growing Porous Glass in the Through-Holes.

Another method for producing an array of through-holes containing aporous material is to machine an array by an appropriate method such asEDM and then to grow material in the array. Porous glass can beintroduced into the array by using pressure to force a mixture ofpotassium silicate mixed with formamide into the array and then bakingfor several hours. An array of capillaries that is fused or embedded ina binding agent can be filled with porous glass by this method and thensectioned with a cooled sectioning saw to create multiple, thin platensof porous-glass filled through-hole arrays. By including particles suchas porous silica or polymer beads in the potassium silicate mix, theaffinity properties and porosity of the material can be adjusted asdesired.

III. Methods of Loading Array Devices with Samples.

Synthesis of an Array by Masking.

Certain methods feature applying at least one mask to a platen, or analigned stack of such platens, such that, when a reagent is applied tothe mask, the reagent communicates with only the through-holes selectedby the mask. In particular embodiments, the reagent can be selected froman aqueous solution, an organic solution, a dry powder, a gel, a gas, oran electromagnetic radiation (e.g., heat, light, X-rays, ultravioletradiation, a magnetic field). Methods for introducing reagents throughthe mask and into the array include applying a mechanical or opticalpressure to the reagent reservoir, diffusion, and electrophoresis.Measures to prevent cross-contamination of neighboring wells includeplacing a second, identical mask on the face of the platen opposite theface to which the reagent is applied, and using a blotter to absorbexcess liquid flowing through the through-holes. The masks and arrayscan be held together by electrostatic, magnetic, or gravitationalforces, or by applied pressure (e.g., by applying a clamp to theperiphery of the stacked platens). Masks and arrays can be separatedwhen liquid fills the through-holes by application of electrostatic,magnetic, or gravitational forces, or by application of a negativepressure to the stacked platens. Stacks of platens can optionally bedried to facilitate separation.

The liquid samples in the through-holes can be subdivided by placing anempty array beneath a filled array or array stack, and then applyingpositive or negative pressure to force a portion of the liquid into theempty array. A small (typically less than 100 μm) air gap is maintainedbetween the filled array and the empty array to facilitate the physicalseparation of the two arrays after the fluid transfer is complete.

Through repetitive cycles of adding masks, introducing reagents, andoptionally washing the array, a defined pattern of chemicals can becreated in the through-holes of the platen by using solution phasechemistry.

By first derivatizing the inner surfaces of the through-holes with alinker molecule that contains a free functional group, the inner surfaceof each hole can be coated with a member of a library of molecules. Thepatterned through-hole array is then used to analyze chemicalinformation. As described above for solution phase systems, repeatedlyadding masks, introducing reagents, and washing the array, can result ina defined pattern of chemicals attached to the linker molecules in thethrough-holes. FIG. 1 is an illustration of this process. A platen (1)having through-holes with derivatized inner surfaces (4) is brought intocontact with a mask (2) configured such that only select through-holesin the platen communicate with the reagents to which the masked platenis exposed. In this embodiment, two identical masks (2,3) are placed incontact with the top and bottom surfaces of the platen so as to preventfluid from entering the covered through-holes. Preferably, the masks andplaten are flat and polished (e.g., to an optical finish) so that theycreate an airtight seal when contacted. Alternatively, the mask orplaten, or both, can be coated with a soft polymer or gasket-formingmaterial to facilitate sealing, or the mask and platen surfaces can bemanufactured with contoured surfaces such that one fits into the otherto form a large contacting surface area. The contoured surfaces can alsoprovide alignment features that can aid in co-alignment of through-holesin the masks and platens. One example of an interlocking array design isshown in cross-section in FIG. 2, where the through-hole arrays arecontoured to have opposing and matching geometrical features. Therequisite geometrical features can be obtained, for example, bypatterned chemical etching or micromilling.

Similar approaches can be applied to the manufacture of a system havingtwo masks with a platen sandwiched in between. The mask-platen sandwichcan then be loaded with a reagent, such that the reagent enters into theopen (i.e., non-masked) through-holes. After the reagent has reactedwith the linker molecules located inside the through-holes, the excessreagent can be washed from the sandwich, and the process can be repeatedwith a new reagent. The mask can then be removed and a new mask applied,or the synthesized material can be removed from each through-hole.

Another embodiment uses only one mask to block one end of thethrough-hole with an airtight seal. The air trapped in the through-holeprevents liquid from entering into the through-hole.

In another embodiment, a porous material derivatized with a linkermolecule having a free functional group is in each through-hole andmembers of a library of chemical probes are attached to the linkermolecules. Alternatively, the inner surfaces of the through-holes aremade porous (e.g., by etching with hydrofluoric acid), and the libraryof probes is attached. Containment of probes within the porous polymeror surface (i.e., as opposed to immobilization of library members on theinner surface of the through-hole) increases the density of librarymolecules in each through-hole.

For chemical synthesis, a porous polymer derivatized with a linkermolecule having a free functional group can be inserted into thethrough-hole. By repeatedly adding masks, introducing reagents, and,optionally, washing the array, a defined pattern of chemicals attachedto the linker molecules can be created in the through-holes. Containmentof linker molecules within the porous polymer increases the density ofsynthesized molecules in each through-hole.

A similar synthetic procedure can be used with a molecular libraryimmobilized inside a porous through-hole surface.

Mask Production Methods.

An embodiment of the invention provides for producing a mask by use of athrough-hole array substantially identical to the array used forsynthesis or analysis. In one embodiment, a mask can be fabricated frommetal, dielectric (glass, polymer) or semiconductor (e.g., silicon,germanium, gallium arsenide). Inertial drilling is a suitablemanufacturing process for fabrication of masks in polymers, glass ormetals. Patterned chemical etching processes, such as deep reactive ionetching (DRIE), provide another suitable manufacturing process forfabrication of masks in semiconductors and dielectrics such as glass.Electrospark discharge machining (EDM) is another suitable manufacturingprocess for fabrication of masks in conductive materials (e.g.conductive semiconductors and metals).

Another embodiment of the invention, shown in FIG. 4, provides forproducing a mask by use of a through-hole array substantially identicalto the array used for synthesis or analysis. A solution (1) is added toeach hole of a through-hole array (2) such that the solution contains amolecule or mixture of molecules that polymerize upon irradiation (Step1). The solution could be for example, an aqueous solution of polymerand a photo-reactive molecule that produces free-radical initiators ofpolymerization when irradiated with ultraviolet light (3) (Step 2). Anexample of a UV curable polymer suitable for mask fabrication can befound in the class of UV-curable polyurethane epoxies. By shiningultraviolet light onto each hole that the artisan wishes to be blockedin a given step of adding reagent to a through-hole array, a mask (4) isbuilt (Step 3). The resulting polymer is impervious to the fluids towhich the mask is exposed. The through-holes to be blocked can beilluminated through an optically opaque mask or illuminated sequentiallywith focused light.

Another embodiment, shown in FIG. 5, uses an array of pins or posts tomake a mask. The external dimensions of the static pin array areselected such that they have a precision fit into a matchingthrough-hole. Viewed in cross-section, a pre-fabricated pin array (1)selectively blocks those through-holes from communicating with a reagentas part of a synthesis sequence. A through-hole array (2) is preparedwith the inside surface of the through-hole derivatized with a linkermolecule having a free functional group (Step 1). A pin array isinserted into the through-hole array (Step 2) and by the process ofintroducing reagent and optionally washing the array, a defined patternof chemicals in the open through-holes is created attached to the linkermolecules (Step 3). The pin array mask is removed and the process isrepeated with a different mask and reagents resulting in a definedpattern of chemicals created in the through-hole array (3) attached tothe linker molecules (Step 4).

Pins in the array are fabricated to precisely fit into each matchingthrough-hole forming a hermetic seal. Polymer coatings (e.g., Teflon)can be applied to each pin to facilitate sealing. An advantage of thisapproach is that the post arrays are reusable and need only to be madeonce. The large contact area between the pin and through-hole interiorsurface ensures a viable hermetic seal. As opposed to the plate masks,only the pins contact the array plate, thereby facilitating decouplingof the mask from the array plate after completion of a synthesis cycle.A further advantage is gained by application of only one pin array toseal through-holes in an array. This is achieved by physical blockage ofthe hole by the inserted pin or by the pressure of air entrapped in thethrough-hole. The pin array can be manufactured by a variety offabrication techniques. One example is to electro-spark dischargemachine (EDM) a regular array of pins having the precision crosssections needed to hermetically seal a through-hole. With a die-sinkingEDM, selected posts could be machined away with a die to form thespatial pattern of pins matching a particular mask configuration. Asecond example is to start with a plate having holes in the spatialpattern matching a mask configuration. The pins are fitted into eachthrough-hole and simultaneously soldered in place.

Another embodiment uses an actuated pin array forming a mask tohermetically seal selected through-holes in an array. The actuated pinarray is similar in design to the static pin array except that each pincan be extended or retracted such as to reconfigure the pin array tomake a different mask. This is different from the static pin array inthat each different mask requires a different pin array. Extended pinsare inserted into through-holes whilst retracted pins are not.Individual pins in the array are electronically addressable to beactuated by one of several types of methods including: piezoelectric,electromagnetic (solenoid), magnetostrictive, shape memory alloy orconducting polymer. One advantage of this approach is a single pin arraycan be reconfigured to produce n!/(n−2)! number of different masks wheren is the number of pins in the array. A second advantage is generationof different masks in an automated manner. This is important when theprocesses requiring masking of the through-hole array are alsoautomated.

In still another embodiment, the mask has holes that allow reagents toflow to selected positions in the through-hole plate. In otherpositions, the mask includes raised features (e.g., pins or bumps) thatfit into the holes to be blocked. This approach aids in alignment of themask and platen, allows a single mask to be used, and ensures a goodseal between the mask and the platen.

The through-hole array can also be fabricated with valves on one side ofthe through-hole array. Each through-hole thus has a valve that eitherblocks or unblocks one end of the through-hole. Pressure of airentrapped in the through-hole prevents liquid from entering the open endof the blocked through-hole. The valve can be formed as a bilayeractuated by shape memory alloy, electrostrictive, electroporous,piezoelectric, magnetostrictive, or conducting polymer materials.Microsolenoid activated valves can also be used to perform a similarfunction.

Another method for producing a flow-mask is to laminate a platen on atleast one side with a non-permeable membrane such as an adhesive tape.The mask is then created by selectively perforating the laminatematerial. Methods by which the laminate can be perforated are: by anactuated pin array, by laser machining, by contacting with a platen thatallow heat to be applied in a localized manner, thus melting or burninga hole in the laminate. Serial dilutions can be performed duringloading.

This operation can be performed to fill a series of through-holes withdifferent concentrations of the same solute. In a typical example, amicrosyringe, or other fluid transfer device, is positioned over thefirst through-hole and used to fill the first and second through-holeswith a 16× solution of the solute. The outer surface of the syringe tipand the faces of the array must be nonwetting toward the solution beingdispensed. For each hole, a sufficient volume of solution, referred tobelow as “Y nl,” is dispensed to overfill the hole enough to createpositive menisci. The microsyringe tip is then rinsed three times andfilled with solvent. The syringe tip is positioned above the secondthrough-hole and Y n1 of solvent is expelled such that it forms adroplet at the end of the syringe tip. The syringe tip is lowered untilthe solvent droplet contacts the solution surface, causing the twoliquids to mix and produce an 8× solution. The syringe plunger is thenwithdrawn to suck up Y nl of 8× solution and dispense it into the nextthrough-hole. Another droplet of solvent is then formed and the processcan be repeated to dispense 4×, 2×, 1×, etc. into individual arraythrough-holes.

Serial Array of Masks on a Flexible Sheet

Since synthesis of an array of probes in through-holes can involve theuse of many masks, a rapid and automated method for interchanging masksis desirable. One method involves creating the multiple masks in asingle, flexible tape, such as a metal or plastic tape with a widthgreater than that of the array to be synthesized. The first mask can bealigned with the array and reagents can be transferred to the array. Thetape can then be advanced to reveal the next mask prior to addition ofthe second reagent. This process can be repeated until the solid-phasesynthesis is complete. For steps that require washing the entire array,a single large hole, or holes corresponding to each position in thearray, can be produced on the tape. For additional through-put andcustomization of the synthesis, the tape can be produced concurrentlywith the synthesis, for example, by having an array of punches or amicro-positioned laser drilling system to create holes as the tapeadvances. A second tape or blotter can be used on the opposite side ofthe array (e.g., to prevent cross-talk). Various methods for aligningthe masks with the arrays can be used, including placing precisealignment notches in the tape, and using optical or amperometricdetection to determine mask position relative to the array.

Synthesis of Arrays Using Masks and a Membrane.

The mask-synthesis methods described here can also be used withnon-addressable porous membranes (e.g., a filter), instead of with therigid platen.

Capillary Tube Array

Viewed in cross-section, a capillary tube array (FIG. 6) is constructedfrom capillary tubing (1) with an external diameter that fits preciselyinto the through-holes of a second array. The tubing array (3) isdesigned such that tubing at one end has a center-to-center spacingequal to the spacing between holes in a through-hole array and tubing atthe opposite end has a center-to-center spacing equal to thecenter-to-center spacing of wells in a microtiter plate (2). Plates withthrough-holes having these separations serve as jigs (4) to hold thetubing in a regular array. Additional through-hole plates placed betweenthe two ends are spacer jigs providing additional support for the tubingarray as the center-to-center spacing is changed over the tubing length.

The internal volume of each tube in the array is slightly greater thanthe total volume of a column of aligned holes in the array stack. Forexample, if the through-hole dimensions in the array are 250 μm×250μm×1000 μm giving a volume per through-hole equal to 62.5 nl, then thevolume of one set of holes in a stack of 100 arrays is 6.25 pl (100×62.5n1). Capillary tubing with an internal diameter of 200 μm and anexternal diameter of 245 μm is readily available; thus a minimum tubelength of 200 mm stores the volume of fluid needed to fill this set ofthrough-holes.

One end of the tubing array is inserted into the wells of a microtiterplate where each tube is inserted into a matching well. A negativepressure is applied across the length of tubing, drawing liquid fromeach well into its corresponding tube. Negative pressure can be appliedto each tube individually or as shown in FIG. 6, the ends of the tubearray can terminate in a chamber that can be partially evacuated. Afterfilling each tube of the array, the microtiter plate is removed. Theliquid can be stored in the tubing array for an indefinite period oftime, either frozen or in a humidified environment. Multiple tubingarrays can be filled from the same microtiter plate (assuming there issufficient volume of liquid per well) or different tubing arrays can befilled from different microtiter plates.

Transfer from a Microtiter Plate with an Array of Flexible Members

As illustrated in FIG. 7, fluid can be transferred from individual wellsof a microtiter plate (3) with an array of flexible members (2) (e.g.,shape memory alloy fibers). The fiber diameter is equal to or less thanthe inside dimension of the through-holes in the array (1) into whichfluid will be transferred. The number of fibers in the bundle can, forexample, be equal to the number of wells in the microtiter plate. Theends of the fibers at one end of the bundle can have a center-to-centerspacing equal to the spacing of the holes in the through-hole array,while the ends of the fibers at the opposite end can have acenter-to-center spacing equal to the spacing of wells in the microtiterplate. The fibers can be held in place with a series of through-holejigs designed to increase the spacing between fibers from one end of thebundle to another. Once fixed in place, shape memory alloy fibers can beheated above their critical transition temperature to make the imposedfiber curvature permanent. After they are cooled to room temperature,the fibers can be removed from the holding jig, with the change in fibercenter-to-center spacing intact. The close packed end of the fiberbundle can then be inserted into the through-hole array into which fluidfrom each well in the plate is to be placed. The opposite end can bearranged such that each fiber is positioned above a well in themicrotiter plate, and the ends of the fibers can be immersed in thefluid contained in each well. On retraction, a small volume drop (4) canremain attached (e.g., by surface tension) to the end of each fiber. Aforce can be applied to the opposite end of the fiber bundle to pull thebundle through the holes of the through-hole array, such that the fluidis brought into contact with the corresponding through-holes. As thefibers are pulled through the hole, surface tension can act to hold theliquid in the through-hole as the fiber is removed.

Pressure Loading

The array can also be loaded by applying a pressure across the platen,thereby causing a dilute solution of reagent and/or sample to flowthrough the array of through-holes. This method can be advantageous ifthe through-holes are already loaded with reagents, and a reaction witha second set of reagents is desired.

Bead Loading

Bead loading can be used to load an entire combinatorial libraryimmobilized on microscopic polymer spheres of uniform size. In thismethod, the through-holes in an array can be shaped so as to hold onlyone microsphere per through-hole. The through-holes can additionallyhave a tapered cross-section, such that the microspheres sits in theholes either at or below the array surface (FIG. 8).

Transferring Contents from a Second Array Replicating a platencontaining through-holes reproduce an array wherein each channelcontains a colony of cells having a unique genetic profile. The masterplate is prepared from a suspension of cells of diverse geneticcharacters. The suspension can be diluted such that when an array isloaded from the dilute solution, an average of one cell is transferredinto each channel. The array is then incubated in an enclosed humiditychamber with the appropriate temperature and agitation for the cell typeuntil the cells have reached mid log phase. The cell number density canbe estimated by observing select channels under an optical microscope,measuring the amount of scattering when light is incident on the arrays,or if the cells also contain a gene for green fluorescence proteinproduction, by measuring the intensity of fluorescence from eachchannel. Various methods can be used to transfer a portion of the cellsfrom each channel into corresponding channels in a second array.

One method of transfer involves freeze-drying the contents of thethrough-holes. A master through-hole array is prepared from a suspensionof cells of diverse genetic characters as described above. A secondidentical through-hole array is filled with growth media. The twothrough-hole arrays are aligned and stacked to mix contents ofcorresponding through-holes. The stacked through-hole arrays arefreeze-dried and separated. The colonies are reconstituted by fillingthe array with media. In the case of robust cells such as bacteria andyeast cells, dehydration by evaporation can be sufficient to remove theliquid without significantly compromising cell viability. This method isalso useful for storing compound libraries such as small moleculelibraries in a dry form. For example, the crystalline compounds canadhere to the walls of the channels. Compounds can then be stored forlong periods of time and reconstituted by the addition of solvent.Compound libraries can also be stored in a powdered or crystalline formwhile frozen in an inert matrix. A suspension of crystalline compoundscan be made in a low molecular weight perfluorinated hydrocarbon andstored frozen. An example of a perfluorinated hydrocarbon that can beused for this purpose is perfluorohexane, which has a melting point of−4° C. and a boiling point of about 59° C. Upon retrieval of the sample,the hydrocarbon can readily evaporate at atmospheric pressure or underan applied vacuum; the samples can then be reconstituted with DMSO,water, or other solvent.

Transferring/Mixing Samples in a Through-Hole Array with Samples on aFlat Surface

Liquid samples contained in a through-hole array can be transferred inpart or in whole to a flat surface having a pattern of hydrophilic andhydrophobic regions. The hydrophilic regions must be spatially isolatedfrom one another and must match the spacing of the through-holes suchthat when the array is contacted with the surface, the contents of eachthrough-hole contacts at most one hydrophilic region. The hydrophobicregions on the surface also serve to isolate the transferred fluids fromone another.

The surface may support an array of samples that can be registered withan array of probes contained in a through-hole array. The samples mustbe spatially isolated from one another and must match the spacing of thethrough-holes such that when the array is contacted with this surface,the contents of each through-hole contacts at most one sample. Thesurface can also contain a hydrophobic pattern matching the pattern ofthe through-hole array to prevent cross-contamination after the surfaceand array are contacted. If a hydrophobic pattern is not provided, theplaten and surface can be pressed tightly against one another to form ahermetic seal and thus prevent mixing between adjacent samples.

Alternatively the flat surface can support an array of probes, such asfluorescently labeled oligonucleotides, chemical substrates, or cells,matched to a through-hole array containing samples. The probes can beattached to the surface in a variety of methods: they can be chemicallyor physically absorbed on the surface, trapped in a porous matrix,attached with an adhesion layer, or contained in a drop of liquid. Theprobes can be used to generate a change in a detectable physicalproperty of the sample (such as fluorescence, optical absorption ormass) in response to a chemical or biological characteristic of thesample as binding activity or enzyme activity.

Plunger Sterilization

Plunger sterilization can be an important aspect of a serial samplingscheme. One approach is to have at least two plungers—while one plungeris sampling, the other is being sterilized. The two plungers can belocated in a common mechanical housing, for example, mounted to rotateabout an axis parallel to the plunger axis. Plunger sterilization can beaccomplished by heat or exposure to sterilizing agent (e.g., 70%ethanol). A wire (e.g., platinum) loop inside a ceramic sheath is anexample of a suitable plunger design. The ceramic sheath impartsmechanical rigidity and is an electrical insulator whereas the wire looppermits heating with an electrical current. As an example, assume that aplatinum wire loop is used, having a specific heat of 4 J/kg-° C. Toelectrically heat a 10⁻⁴ kg wire to 1000° C. in 0.2 s requires a maximumcurrent of 4.5 A, which is easily achieved with medium power thyristors.Rapid cooling can be achieved from the spray of a volatile sterilizingagent (e.g., ethanol), the high latent heat of vaporization of which canaid in cooling the heated wire. Alternatively, the wire can be rapidlycooled by a spray of gas or liquefied gas such as liquid nitrogen.

Sequential sampling with a single sampling device can be extended to alinear array of sampling devices as, for example, a linear array ofmechanical plungers. There can be a substantial time saving (e.g., 1/Mfor an M×M array of through-holes), since motion along one orthogonaldirection can be avoided. Similar time saving considerations are appliedto two-dimensional sampling techniques. In an alternate approach,pressure generated by spatially localized jets of liquid, solid, or gascan be used in place of the plungers.

Loading with High Protein/Surfactant Media

Loading the platen by submerging the platen into a reagent of interestwhen the reagent or media is high in protein or surfactant and thereforelow in surface tension, can be a challenge. Droplets of the low surfacetension fluid can remain on the surface of the platen after removal fromthe fluid to be loaded. If droplets or a surface coating of protein richmedia remains on the surface, it can result in contamination of theassay or crosstalk between through-holes. This problem is significantlylessened by pulling the platen up through a layer of a hydrophobic fluidthat is immiscible with the fluid to be loaded. This provides a wipingor “liquid squeegee” effect, removing the proteins or surfactantsadhering to the surface of the platen. The wiping fluid setup can begenerated one of at least three methods.

Submerging the platen in the fluid to be loaded, ensuring thethrough-holes are filled. The platen is withdrawn from the fluid to beloaded, and submerged in a hydrophobic fluid that has a greater affinityfor the proteins or surfactants than the protein or surfactant has forthe surface of the platen. Such fluids can include but are not limitedto perfluorodecalin, silicone oil and mineral oil.

Alternatively, the platen is first submerged into the fluid to beloaded. Then a small amount of a less dense, hydrophobic fluid such asbut not limited to mineral oil and silicone oil is gently layered on thesurface so that the surface of the loading fluid is completely coveredwith this wiping fluid. Then the platen is slowly removed from theloading fluid, up through the wiping fluid.

Additionally, a container can be used as in FIG. 23. The container has abaffle that extends from one side if the container to another, but doesnot extend to the bottom of the container. The container is filled to alevel midway up the baffle with the fluid to be loaded. This creates twoopen surfaces of loading fluid connected by a channel underneath. Wipingfluid is gently layered on one of the surfaces, and is kept from theother surface by the baffle.

The platen is submerged into the fluid to be loaded, underneath thebaffle, and removed through the wiping fluid. This method for employingthe wiping fluid is well suited for high throughput and automationmethods.

Synthesis of Arrays by Selective Loading of Fluids into Through-Holes.

An array of pins (1) can be fabricated with pins arranged to beco-registered and co-aligned with a second regular array ofthrough-holes (2) (FIG. 3). Each through-hole can be prepared such thatlinker molecules suitable for chemical synthesis are immobilized on theinterior surface of each through-hole. The ends or tips of the pins canbe made hydrophilic over a pre-determined surface area whilst theremainder of the array surface area is made hydrophobic. In oneembodiment, the tips of the pins in the pin array are brought intocontact with the fluid to be loaded into the through-holes of thethrough-hole array (3). When retracted, a small volume liquid dropadheres to the hydrophilic region of each pin (e.g., by virtue ofsurface tension). The volume of liquid adhering to the pin is determinedby the relative surface energies between the liquid and solid surfaceand the depth of immersion of the pin into the liquid relative to thehydrophobic surface area. The pin array with the adherent liquid dropscan be arranged relative to the through-hole array such that thethrough-holes into which liquid is to be placed are aligned relative toeach pin with an adherent liquid drop. The two arrays can be broughtinto contact such that the drops enter into the correspondingthrough-holes (e.g., by capillary pressure). Removal of the pin arrayleaves behind the fluid placed into the through-holes of the array (4).A chemical reaction can thus be initiated between the linker moleculesimmobilized on the interior surface of the through-hole and the liquidplaced in the through-hole. The chemical reaction rate can be increasedby raising temperature and/or changing the partial pressure and/orcomposition of gas in the atmosphere surround the through-hole array.After the reaction is complete, the array can be washed to removeunreacted components, and dried to remove excess solvent. A second pinarray with either the same or different pin configuration can be loadedwith fluid and the synthesis process can be repeated. Because the arrayloading process can rely on simple mechanical motions, the array loadingcan be quite rapid. The rate-limiting step, therefore, would be thesynthesis step itself

An alternative embodiment features the use of a pin array sparselypopulated with pins aligned with respect to the through-holes of aregular array. The pins can be fabricated such that their length is atleast twice the thickness of the through-hole array platen. Eachthrough-hole can be prepared such that linker molecules suitable forchemical synthesis can be immobilized onto the interior surface of eachthrough-hole. The end of each pin can, for example, by made hydrophilicand the remainder of the array can be made hydrophobic. The lateraldimension of the pins can be set such that the pins can be inserted intothe matching through-holes in a regular array. The pin array can then beinserted through the second through-hole array such that the pins extendthrough to the opposite side of the platen. This assembly can bearranged relative to the surface of a fluid that is to be placed intothe through-holes through which pins have been inserted. The tips of thepins can be brought into contact with the fluid surface, and, onretraction, small volume drops can adhere to the end of each pin. As thepin array is retracted relative to the through-hole array, the liquiddrops can come into contact with the through-hole into which the pin hasbeen inserted. As the pin leaves the through-hole, surface tension cankeep the fluid volume inside the through-hole.

An advantage of both embodiments is that they provide a rapid, simple,and precise method by which fluid can be loaded into through-holes of anarray. Fluids containing surfactants can, for example, be easilytransferred into the array with minimal contamination between adjacentthrough-holes because of the long path along the array surfaceseparating the ends of adjacent pins.

Synthesis of a Stochastic Array.

A stochastic array can be created using a nozzle moving randomly todifferent through-holes on the array. Loading the through-holes in astochastic method, wherein one variable of the sample is varied in arandom manner has many applications. For example, the stochastic loadingcan vary with respect to the concentration of a particular reagent inthe sample loaded. The difference in concentration of the reagent allowssimultaneous sampling, in a controlled manner, of many differentreaction conditions. For example, this sort of application of samplescan be used to optimize reaction conditions in chemical synthesis or canoptimize parameters of a crystallization experiment.

IV. Reactions/Experiments in the Platens.

Synthesis of Combinatorial Libraries.

The present invention provides new methods for producing a combinatoriallibrary in a platen. The types of combinatorial libraries that can beproduced using the new methods include, but are not limited to, nucleicacid arrays, peptide arrays, protein arrays, polymer arrays, and arraysof small molecules.

Certain of the new methods include immobilizing a linker molecule on theinner walls of the platen's through-holes, or in a porous materiallocated inside the through-holes, and sequentially flowing reagentsthrough masks to build a pattern of chemicals. For example, to create anucleic acid array, phosphoramidite monomers can be sequentially placedin the through-holes in defined patterns with activation, reaction,washing, and deprotection steps in between each addition of monomer. Tocreate an array of small molecules using solid or liquid phase syntheticchemistry, linker molecules with, for example, protected amide groupscan be sequentially placed in the through-holes in defined patterns withwashing and deprotection steps between each synthetic reagent additionstep. Chemical synthesis with solid phase chemistry can be carried outon core molecules linked to the interior surface of a through-hole,inside a porous material placed in a through-hole, or on a polymer beadplaced in the through-hole. Core molecules with chemically active sidegroups can also be prepared in the through-holes using solution phasechemistry.

Chemical and Physical Process Optimization

Optimization of a chemical or physical process requires searching amultivariate space of experimental conditions for a subset of thoseparameters producing the desired outcome. The search strategy can beeither systematic or stochastic; either or both strategies can beimplemented as embodiments of the present invention. Systematicoptimization is aided by producing an array of chemical or physicalconditions from one through-hole to the next in a known and regular way.

Stochastic variation of reagent concentrations from one through-hole tothe next can be accomplished, for example, by first uniformly loading anarray with a first reagent. A container holding a second reagent canthen be positioned above the array, for example, on a motorized two-axismount, and the second reagent can be dispensed through a nozzle with anelectronically controlled valve. The nozzle can be moved to differentrandomly selected array positions (or to positions determined by analgorithm), and the amount of liquid dispensed through the nozzle can bedetermined by a randomly selected (or algorithm-selected) time durationless than a pre-selected maximum. In this manner, different amounts ofthe second reagent are dispensed into through-holes containing the firstreagent. This process can be repeated with additional reagents asneeded. The reactions with optimal outcomes can be identified byanalyzing the contents of the through-holes. If the second reagent isdistributed randomly, rather than according to an algorithm, lack ofspecific knowledge regarding the starting conditions for these reactionscan make duplication difficult. However, if one is interested in thereaction products alone, then a stochastic approach can provide a facilemethod for rapidly searching a large experimental parameter space for adesired reaction outcome. Moreover, the reaction conditions cansometimes be inferred, for example, by examining the contents of otherthrough-holes in the array where little or no reaction occurred, andthen combining the results in a multivariate plot. Optimal reactionconditions can be inferred from domains containing little or no data.Another approach to assess initial reaction conditions is to produce areplica plate using the same dispensing protocol but into an arrayuniformly loaded with solvent without any of the first reagent.Comparison of through-holes showing the desired chemical activity withthe contents of the corresponding through-hole in the replica platewould provide information as to the most likely starting conditions ofthe observed reaction.

If large numbers of conditions need to be tested, the multiple reagentscan be randomly sprayed onto the array until all of the through-holeshave been filled. The surface can then be wiped with a rubber spatula toremove excess fluid. A reaction can be initiated by stacking the arraywith a second array, and the result can be probed optically. Because thecontents of each address in the array will be unknown, one can eitherchose promising addresses, and then analyze the contents to determinewhat was in the hole, or else replicate the plate prior to initiatingthe reaction, and then use the replicate plate to determine the optimalconditions.

These examples also allow for physical parameters to be eithersystematically or randomly varied from one through-hole to the next. Forexample, a temperature gradient can be imposed across one ortwo-dimensions of an array fabricated from thermally conductivematerial, for example, by holding the edges at different temperatures.If the temperature of a heating/cooling source is changed with time,then the temperature distribution across the array can be varied. Therate of temperature change is generally proportional to temperature;thus, both the temperature and the rate of temperature change can varyfrom one through-hole to the next. Alternatively, a focused laser beamcan be directed to heat each through-hole independently, thus enablingcontrol of the liquid in each through-hole with time as described, forexample, in U.S. Pat. No. 5,998,768, incorporated by reference in itsentirety.

Protein Crystallization

X-ray diffraction from crystallized proteins is an important analyticaltool for determination of protein structure and function. Proteins canbe difficult to crystallize because they generally include amultiplicity of hydrophobic and hydrophilic molecular groups. As aconsequence, proteins often crystallize only under a specific set ofsolvent, pH, salt concentration, and temperature conditions. A highthroughput method for protein crystallization is enabled by the presentinvention.

Screening Methods

The parameters that determine whether or not a biochemically efficaciouscompound is suitable for further development as a pharmaceuticalcompound include absorption, distribution, metabolism, excretion, andtoxicology (“ADMET”). As the number of potential drug leads increasesdue to advances in primary high throughput screening, ADMET testing canbecome increasingly rate limiting in the drug discovery process.

Adsorption.

Oral administration is the preferred route of administration for smallmolecule drugs. For an orally administered drug to have biologicalefficacy it must be bio-available (i.e. it must have the ability to passthrough the gut and into the bloodstream). The ability to assess theability of a drug candidate to pass through the lining of the gut in anin vitro assay is highly desirable. Typically, such absorption assaysutilize a monolayer of cells grown on a semipermeable membrane thatseparates two liquid-filled chambers. The drug candidate is added to oneof the chambers and after sufficient time for diffusion or transport,the concentration of the molecule in the other chamber is quantitativelyanalyzed.

One such absorption assay commonly used in the bio-pharmaceuticalindustry is the CaCo-2 absorption assay. The CaCo-2 assay interrogatesthe ability of a molecule to pass through a single layer of a colon cellline, known as CaCo-2. Typically, the rate of both apical to basal andbasal to apical diffusion across the cell layer is determined. The CaCo2assay is usually performed in an apparatus that provides two chambers offluid separated by a porous membrane. The membrane is permeable to cellgrowth products, but acts as a support and impermeable barrier for thecells. A monolayer of cells is grown across the surface of the membrane,and the active or passive transport of molecules from one chamber to theother across this cell barrier is assayed.

The platens containing arrays of through-holes can be configured severalways to provide an array of absorption assays (e.g. using CaCo-2 orother cells), thus increasing throughput and minimizing reagent volumes.In one embodiment, a isotropically porous membrane (such as, but notlimited, to a PTFE filter) treated to provide a biologically compatiblesurface for growing adherent cells. The membrane has dimensions at leastthe dimensions of the array of through-holes, and is placed on oneplaten so that it covers the through-holes of the platen. A secondplaten with matching through-holes is placed on the membrane so that themembrane is sandwiched between the two platens. Pressure is applied tothe platens so that the membrane is collapsed between adjacentthrough-holes and no chemical crosstalk may occur between non-opposingthrough-holes, but allowing chemical communication between opposingthrough-holes for the assay.

In a second embodiment, the membrane is anisotropically porous,consisting of parallel pores through the membrane. Examples of this typeof membrane are Isopore and Nucleopore filters sold by MilliporeCorporation. As in the previous embodiment, the membrane is sandwichedbetween two platens, and pressure is applied. In this embodiment,pressure is not required to collapse the membrane between adjacentthrough-holes, but only to seal between adjacent through-holes. Theparallel pores of the membrane allow chemical communication betweenopposing through-holes but not adjacent or non-opposing through-holes.

In a third embodiment, the membrane is patterned with regions ofporosity spaced and sized like the pattern of through-holes in theplaten. The membrane is sandwiched between two platens so that the areasof porosity match up with the through-holes, allowing chemicalcommunication between opposing through-holes but not adjacent ornon-opposing through-holes.

In a fourth embodiment, the membrane is made from a platen ofthrough-holes, in which the through-holes contain a porous material,such as but not limited to porous silica. In this embodiment, themembrane platen is sandwiched between two platens of though holes,allowing chemical communication between opposing through-holes but notadjacent or non-opposing through-holes.

Metabolism.

For metabolism studies, compounds can be tested for their propensity tobe degraded by various cytochrome P-450 (CYP-450) enzymes or by livermicrosome preparations. Propensity for causing drug-drug interactionscan be estimated by assaying inhibition of various CYP450 enzymes byeach drug or drug candidate.

An embodiment of this invention provides for measurement of cellularmetabolism of compounds from a library. As described above in connectionwith adsorption assays, the compounds can be small organic molecules,peptides, oligonucleotides, or oligosaccharides. The cells can either besuspended in liquid, or can be grown as a monolayer of cells inside thethrough-holes or on a membrane having high longitudinal permeability andlow lateral permeability. The membrane can include, for example,polymerized monomers in the through-holes of a plate, orhydrophilic/hydrophobic domains in a flexible membrane with domain sizeand center-to-center spacing equal to that of the through-hole array.Volumes of known concentrations can be loaded from the compound libraryinto one through-hole array. The cell array or layer can be placed incontact with the library array and incubated, and the array compositioncan be analyzed to determine the change in compound composition oramount with cellular metabolism.

Toxicity.

An embodiment of this invention provides for measurement of cellulartoxicity of compounds from a library. As described above in connectionwith adsorption and metabolism assays, the compounds can be smallorganic molecules, peptides, oligonucleotides or oligosaccharides, andcan either be suspended in liquid or grown as a monolayer of cellsinside the through-holes or on a membrane having high longitudinalpermeability and low lateral permeability. The membrane can include, forexample, polymerized monomers in the through-holes of a plate orhydrophilic/hydrophobic domains in a flexible membrane with domain sizeand center-to-center spacing equal to that of a through-hole array.Volumes of known concentrations are loaded from the compound libraryinto one through-hole array. The cell layer can be placed in contactwith the library array and incubated, and the cells in each through-holecan be analyzed for viability.

Ligand Screening by Affinity.

It can be desirable to measure or rank the affinity of various membersof a compound library toward a particular target macromolecule, or tomeasure the affinity of an analyte toward various members of a probearray. Such screening can be carried out using the new methods describedherein. For example, affinity experiments can be carried out byimmobilizing a target in many holes of the through-hole array andprobing with a library of potential ligands, or by immobilizing a ligandlibrary in an array and probing with a target.

Thermal Denaturation Ranking.

As new drug targets are rapidly being discovered, methods are needed tofind molecules with affinity to these targets in the absence of afunctional assay. Fulfillment of this goal can be accomplished byimmobilizing the target biomolecule on the inner surfaces of an array,incubating the holes of the array with a library of compounds, anddetecting those members of the array that retain a compound. Boundcompounds also stabilize target molecules to thermal denaturation to adegree that can correlates with the degree of affinity. By detectingunfolding of protein as a function of temperature or denaturing solventcondition, affinities can be ranked, as described, for example, in U.S.Pat. No. 6,020,141 to Pantoliano et al.

Gene Probes.

An embodiment of the invention provides for the production of athrough-hole array containing numerous, known nucleic acid sequences,adding a nucleic acid solution that has at least some unknown sequencesto each of the holes in the array, providing sufficient time,temperature; and solution conditions for the unknown nucleic acid tobind specifically to complementary nucleic acids in the through-holes,and analyzing the degree of hybridization between the nucleic acids ofknown and unknown sequence in each through-hole. After binding, thearray can be washed with a solution of the desired stringency. Often,the unknown nucleic acid has a fluorescent probe attached. An advantageof the invention is that amplification of the signal can be achieved byusing an enzyme reaction that is associated with the hybridized nucleicacids. For example, the unknown nucleic acid can be labeled withhorseradish peroxidase and incubated with a substrate that produces aluminescent, fluorescent or chromogenic signal upon reaction with theenzyme following the binding and washing steps. Such amplificationtechniques can be incompatible with conventional nucleic acid arrays onplanar surfaces, since the activated substrate in solution generallycannot be assigned to a particular point on the array due to diffusion.PCR or other thermal-cycling reactions may be performed in the array bysubmerging the array in a water-immiscible liquid such as an oil,alkane, or perfluorinated solvent. The array and water-immiscible liquidmay be contained in a thermally conducting container such as a metalbox, and then inserted into a thermal cycler adapted to receive the box.

Long-Term or High Temperature Culture of Cells

In order to minimize evaporation, it is known in the art to layer asmall amount of a low volatility, immiscible liquid on top of a smallvolume of aqueous reaction media. For example, a small amount of mineraloil can be layered on a reaction vial containing a PCR (Polymerase ChainReaction) reaction, in order to minimize evaporation during heatingcycles. It is also known that fluids with high oxygen solubilitycontents can be used to enable oxygen transport to systems that requireoxygen. It is a novel aspect of this invention that an immiscible fluidwith a high oxygen solubility content can be used to eliminateevaporation from the array of sub-microliter samples, while facilitatingoxygen transport to maintain cell viability.

In order to culture non-adherent cells in a nano-volume format for longperiods (e.g., greater than 12 hours), it can be desirable to reduceevaporation by containing the cells in a hydrophobic, low volatilityfluid. To allow for aerobic respiration or other gas exchange process tooccur, an oxygenated emulsion of perfluorinated compounds can be used.These compounds have been the subject of clinical testing as artificialblood substitutes. Examples include perfluorodecalin, which is sold asFluosol-DA™ by Green Cross Corp. of Japan; Oxycyte™, which is beingdeveloped by Synthetic Blood International; and Oxygent™-brandperfluorooctylbromide, which is being tested by AlliancePharmaceuticals. In a typical application of these methods and materialsusing the through-hole array, cells are grown in a platen that issubmerged in perfluorodecalin, while oxygen or air is bubbled throughthe medium in a manner that does not disturb the cells.

Culture of cells that adhere to the walls of the through-holes or toporous substances immobilized in the through-holes is comparativelysimpler, as oxygenated aqueous media can be perfused through or aroundthe platen as required.

Culturing thermophilic organisms under aerobic conditions at low volumesand high temperature can be particularly problematic, since evaporationtends to act quickly at temperatures such as 90° C. By submerging thearray of through-holes in an appropriate fluorinated solvent, thesetemperatures can be used without significant evaporation, whilemaintaining a supply of oxygen to the cells.

Often the act of assay detection or sample “picking” may require that aplaten be exposed to non-humidified environments or bright lights for aperiod of time. These are conditions under which evaporation from thethrough-holes can be problematic. In a typical experiment, sampleevaporation is minimized by performing operations under a layer ofimmiscible fluid such as but not limited to perfluorodecalin, andsamples may be added to or removed from the through-holes with amicrosyringe while submerged under such a fluid. Other operations, suchas imaging and platen manipulation such as platen stacking may beperformed under the immiscible fluid as well.

V. Methods of Analyzing and Manipulating Output from Array Devices.

Methods of Transferring Samples from Through-Holes.

Still another method features transfer into microtiter plates. In orderto recover samples giving a positive response to a test, there is oftena need to transfer fluid from selected through-holes in a high-densityarray plate to a microtiter plate having a lower density of wells.Often, this transfer process must be performed with sterile technique.This will allow for sampling of materials held in through-holes withselected properties from a larger collection of samples. There are threegeneral methods for transferring fluids from the high density arrayplate to the wells of a microtiter plate: transfer with a singlesampling device, transfer with a linear array of sampling devices andtransfer with a two-dimensional array of sampling devices. Samples fromproscribed through-holes can be removed by spatially localizedmechanical action. One general embodiment is to insert a member throughthe hole to mechanically displace the material out the opposite side andinto a receptacle positioned beneath the hole. A second generalembodiment is to apply a localized gas or liquid jet to cause materialin the hole to be displaced out the opposite end and into a receptaclepositioned beneath the hole. A third, but slower, method is to transferliquid from the hole to a waiting receptacle by transferring liquid ontoa pin or into a syringe, moving the pin or syringe to the receptacle anddispensing the liquid.

In order to maximize throughput of the transfer system, the number oftimes that the low-density plate is moved should be minimized. If thehigh-density array is imaged and the imaged stored on a computer, thecoordinates of the desired through-holes is available for input into thetransfer apparatus. By aligning the high-density array above thelow-density plate, and calculating which desired samples sit above anempty well in the low-density plate, a maximum number of samples can betransferred without re-positioning the low-density plate. Thelow-density plate can then be moved to a position that allows thegreatest possible number of samples to be transferred in the next step.In order to minimize the cost of the transfer apparatus, thehigh-density through-hole array should remain stationary to avoid use ofa high-precision alignment system. Some high-throughput systems that canbe used in this way are described below.

Another method features transfer with a single sampling device. Thismethod can be accomplished, for example, by fast sequential positioningof a mechanical plunger over the through-holes to be sampled and pushingthe plunger through the hole to transfer the hole's contents to the wellof a microtiter plate located at a small distance below the through-holearray (6) (FIG. 9). The mechanical plunger (1) can be actuated by, forexample, a linear electromagnetic motor (2). This process is repeateduntil each well (3) of the microtiter plate (4) contains the contents ofa different through-hole. Once complete, the filled plate is replacedwith an empty plate and the process is repeated. A servo-controlled,linear motor-actuated two axis stages (5) with magnetic or air bearingscan position a mechanical plunger whose diameter is slightly less than athrough-hole diameter to within < 1/100 of a hole diameter (˜1 μm) withvelocities up to 1 m/s. Thus if 1% of a 100,000 hole array is to betransferred to microtiter plates and the time to sample each hole is onaverage 0.2 s, then 1% of the array can be sampled in about 200 s. Thistime excludes, of course, the time required to change microtiter plates,the time needed to sterilize the plunger between samples and, if needed,the time to position a well below the sampled through-hole.

Spatially Localized Gas, Liquid, or Solid Jets

With reference to FIG. 10, a beam from a laser (1) passes through ashutter (2) and can be directed by a beam scanning system (3) to befocused onto a specified through-hole (4) in a high-density array ofthrough-holes (5). The laser wavelength can be chosen to coincide withan absorption band of a fluid in a targeted through-hole. Thecorresponding absorption coefficient can be such that a large percentageof the incident laser radiation is absorbed in a thin layer at the topof the liquid column. The shutter can control the length of time theliquid in the through-hole is exposed to laser radiation. When theshutter opens, laser light illuminates the liquid, and sufficient energyis absorbed during the exposure time to rapidly heat a thin liquid layerto vaporization, causing the rapid build-up of pressure at one end ofthe through-hole. The resulting force from the expanding vapor causesejection of liquid from the opposite end of the hole (6) into a well ofa microtiter plate located below the through-hole array (7). A furtherincrease in force is possible if the volume above the heated surface ishermetically sealed thus increasing the pressure applied to the liquid.Rapid vaporization and expulsion of liquid from the column requires thelaser energy to be deposited in a time less than the thermalizationtime. Rapid expulsion is needed to increase throughput and to preventsubstantial degradation of the cells or reagents contained in theliquid.

The case of a water-filled through-hole provides an illustrativeexample. Indeed, in many cases, the analytical substance is in water.The absorption coefficient of water at 10.6 μm is −1000 cm⁻¹ indicating99% of the incident radiation will be absorbed within 46 μm of thesurface—a small fraction of the water column's length assuming a lengthof 0.5 mm or greater. The thermalization time, τ, is the time requiredfor the water column to reach thermal equilibrium and is given byτ=l²/4α where 1 is the distance from the source of thermal energy and αis thermal diffusivity (=κ/cφ in which the thermal conductivity is κ, cis the specific heat and ρ is the density. Inputting appropriate valuesfor water, the thermalization time for a column of water 1 mm in lengthis 1.75 s while for a column 0.5 mm long, τ is 0.44 s. Adiabatic heatingwith the focused laser beam will take place if the laser pulse length Δtis less than τ.

The peak pressure generated by the instantaneous vaporization of avolume of water 46 μm thick by 200 μm in extent can be estimatedassuming the water vapor is an ideal gas. The pressure, P, in thisvolume, V (=1.4×10⁻¹² m³), when the liquid is vaporized is P=nRT/V wheren is the moles of water (=88 nanomoles), T is the gas temperature (=373K) and R is the ideal gas constant (=8.2 m³-Pa/mole-° K). Inputtingthese values gives P_(max)=186×10⁶ Pa equal to a force of 5.8 N on thewater column; sufficient to expel the liquid from the through-hole.

The laser power to vaporize a 46 μm thick layer of water in a 200 μmdiameter through-hole can be found by computing the energy, Q, tovaporize this volume of water: The thermal energy is found for Q=m(cΔT+ΔH_(vap)) where m is the mass of the water in this volume (1.6×10⁻⁹kg), c is the specific heat of water (=4184 J/kg/° K.), ΔT is thetemperature change (353° K) and ΔH_(vap) is water's latent heat ofvaporization (=2.3 MJ/kg). Inputting these values gives Q equal to 6 mJ.Assuming 99% absorption of the incident laser energy, 6 mJ is depositedin the sample by a 10 W laser illuminating the liquid surface for 0.6ms. For random-access scanning, typical settling times forgalvanometer-steered mirrors is 10 ms and for 1000 through-holes in anarray to be individually addressed, it will take approximately 10.6seconds.

In alternative embodiments, solids (e.g., powders) or liquids can beused to displace or force out (e.g., under pressure) the contents ofspecific through-holes or the contents of the through-holes in general.(See FIG. 22) Examples of liquids that can be used include liquids thatare miscible with the contents of the through-holes (e.g., water whenthe contents of the through-holes are aqueous) or liquids that areimmiscible with the contents of the through-holes (such as oils ororganic solutions when the contents of the through-holes are aqueous).

Application of pressurized liquid to an array can be used to washcontents of each hole into, for example, a common container.

Explosive Charge.

An extension of the previous embodiment is the expulsion of the liquidfrom a through-hole by a pressure wave generated by rapidly expandinggas on ignition of an explosive charge located in proximity to one endof the through-hole. With reference to FIG. 10, an array of discreteslow-burning explosive charges (1) is co-registered with respect to thethrough-hole array such that one charge is located above onethrough-hole. Each charge is placed in a chamber having a thin membraneas a common wall with the through-hole. The charge array is bonded (ortightly attached to) the through-hole array. Examples of explosivematerial for this application include plastic explosive sheets such as“C4”, or trinitrotoluene (TNT) embedded in plastic. The charge array isaddressable, for example, electrically or optically. An individualcharge can be ignited by passing an electrical current through aresistive element (2) located in the chamber or by the thermal energydeposited in the chamber by a focused laser beam. Once ignited, theexpanding gas from the explosion generates sufficient pressure to burstthe separating membrane and drive liquid (3) from the through-hole (4)into the well (5) of a microtiter plate (6) located below thethrough-hole array (7).

Alternatively, as shown in FIG. 11, the explosive charge can be embeddedas a uniform stochastic distribution in a thin plastic sheet (1).Conversely, the explosive chemicals could be printed onto the sheet inthe same pattern as the through-hole array, as shown in FIG. 12. Printedonto the sheet are resistive elements (2) at discrete spatial locationswith the same pattern as the through-hole array. Alternatively, spatiallocations on the sheet are addressable by a focused laser beam providingthe energy required to ignite the explosive chemicals. The sheet isbonded to the through-hole array (3) and the charges ignited above thethrough-holes whose contents (4) are to be transferred into a well (5)of a microtiter plate (6) located below the through-hole array.

To increase the inertia imparted to the liquid column from the expandinggas charge, metal or ceramic microspheres are mixed with the explosivecharge and are accelerated by the explosion. Alternatively, a plug ofmaterial between the explosive charge and liquid column accelerated bythe explosion will act as a mechanical plunger to expel liquid from thethrough-hole.

Forming the exit of the chamber containing the explosive charge into anozzle will increase the spatial localization and inertia of the exitinggas before impacting the liquid column. The nozzle exit either is fluidwith the chamber surface or protrudes slightly to insert into theopposing through-hole.

Sample Aspiration.

Liquid samples in a through-hole or group of through-holes can betransferred out of the through-holes by aspiration into a tube orchannel. The tip of a piece of flexible or rigid tubing, generallyhaving an outer diameter narrower than the inner diameter of thethrough-hole, can be aligned within the through-hole. Application ofnegative pressure to the distal end of the tubing can then be used toaspirate fluid from the through-hole into the tubing. The amount offluid to be aspirated can be accurately controlled by manipulatingseveral variables. For example, the length and internal diameter of thetube can be determinative of the pressure drop across that piece oftubing, which can in turn affect the rate of flow through that piece oftubing for a given amount of applied negative pressure. A metered amountof fluid can be aspirated from the through-holes into a valve assembly,from which the fluid can be moved by positive or negative pressure toany type of fluidic circuit that is required by a given application. Inone embodiment, fluid is aspirated from a through-hole into a fluidicvalve. Actuation of that valve introduces the fluid via positivepressure to a mass spectrometer for analysis of that fluid.Alternatively, further sample preparation or characterization (e.g.,chromatography, spectroscopy) can be performed on the fluid once it hasbeen aspirated from the through-hole.

Electrophoresis and Electroblotting:

The invention also provides methods for introducing an ionic sample intoa through-hole array containing chemical probes, for modulating thestringency of the binding between the probes and certain ions in thesample, and for removing an ionic sample from the through-hole array.The method includes placing the through-hole array containing chemicalprobes localized in the holes into a buffer in an electrophoresisapparatus. A sample containing ions is introduced into theelectrophoresis apparatus on one side of the planar through-hole array,such that when an electric field is applied, the ions of the appropriatecharge will migrate in the direction of the through-hole array. If aparticular ion does not bind to the array and the electric field isapplied for sufficient time, that ion will migrate through the hole tothe opposite side of the through-hole array. By periodically changingthe direction of the electric field, approach to equilibrium in thebinding between the charged species and the chemical probes in thethrough-holes can be accelerated. Partial purification of the sample tobe analyzed can optionally be achieved by electrophoresis of the samplethrough a gel prior to its migration to the through-hole array. Once theanalytes of interest in the sample have associated with the chemicalprobes, the field can be applied for sufficient time and with sufficientstrength to dissociate non-specifically bound ions from the chemicalprobes. The through-hole array can then be taken from theelectrophoresis apparatus, and the pattern of binding can be analyzed.The bound analytes can also be removed for further analysis, forexample, by electroblotting onto a membrane, and then removing themembrane for further analysis.

Chromatic Analysis of Samples in an Array of Through-Holes.

For many applications, samples must be isolated, purified, orconcentrated by a chromatographic step. Many different types ofchromatography can be performed on liquid samples. Examples ofwell-known chromatographic methods include, but are not limited to, ionexchange, reversed phase, size exclusion, bio-affinity, and gelpermeation chromatography. The chromatography matrix can be in the formof an insoluble bead, gel, resin, polymer, or slurry. The matrix canalternatively be a micro-machined structure. One embodiment of such amicromachined structure is a grouping of square through-holes orchannels with sides of dimension on the order of 0.01 to 10 gm. Thewalls of these through-holes can be coated with a surface having adesired affinity such that a separation is achieved as sample is flowedthrough the group of through-holes. The analyte mixture of interest isthen introduced to this matrix and selective binding of components ofthe analyte mixture to the matrix takes place. Analytes of interest orcontaminants can then be selectively eluted from the matrix by changingthe physical or chemical environment of the matrix.

Liquid chromatography is typically performed in a column in which thechromatography matrix is immobilized and the analyte is flowed throughthe column, allowing chemical and/or physical interaction between thesample and the chromatography medium to take place. The length andinternal diameter of the array of capillaries generally determines theamount of chromatography matrix that can be loaded into each column and,therefore, is directly related to the loading capacity of each column.

Immobilizing a chromatography matrix inside an array of through-holescan create an array of miniature liquid chromatography columns. Asuitable length-to-diameter ratio of the array of through-holes can beselected. Typically, a minimum length-to-diameter ratio of at leastabout 10 is required to form an effective chromatography column. Incertain embodiments, the internal diameter of the columns formed in thethrough-holes is less than a millimeter, allowing for precise andaccurate manipulation of very small amounts of sample. Thechromatography matrix can be immobilized within the chromatographycolumns by positioning a porous frit at the exit end of the column or bychemically binding a porous polymeric ceramic or glass substrate to theinside of the column. The porous ceramic or glass substrate can eitheract as a frit to immobilize a bead or resin chromatography media or as amethod to increase the total surface area within a column. Insurface-effect driven chromatographic methods (e.g., ion exchange,affinity, or reversed phase chromatography), chemical derivatization ofthe interior surface of the columns can provide the necessaryseparation.

In one embodiment, samples are loaded into the array of columns withsyringes. A submicroliter volume of sample can be drawn into the needleof the syringe or a bank of syringes with a spacing co-registered to thespacing of the array of columns, and then transferred to the array ofcolumns. The barrels of the syringes can contain a larger quantity ofliquid for performing wash and/or elution steps in the bundle ofcapillaries. The sample in the needle of the syringes and the liquid inthe barrel of the syringes can be isolated from one another by drawingup a small amount of air into the syringes. The needles of syringes canbe docked into the array of capillaries with a liquid-tight compressionfitting. As the content of the syringes are ejected into the array ofcolumns the samples in the needles of the syringes initially elute ontothe column bed. The chromatography media used and buffer drawn into thebarrel of the syringe will dictate the chromatographic separation. Oncethe samples have been loaded onto the columns, the syringes can beremoved from their docking ports and a second aliquot of a similar ordifferent buffer can be drawn into the syringes. As many wash and/orelution steps as necessary can be performed by re-docking the syringesto the array of capillaries by tightening the compression fitting andejecting the liquid from the syringes. A first array of columns can alsobe mated to a second array of columns for further separation.

It is often desirable to analyze the eluate from a chromatography columnin real time using a variety of spectroscopic methods. Spectroscopicdevices for interrogating the eluate from a chromatography column arewell known. If required by the specific application, the eluate fromeach column in a bundle of columns can be analyzed on-line in real time,spectroscopically (e.g., by absorption, fluorescence, or Ramanspectroscopy), electrochemically, or otherwise. The light from thespectroscopic light source can be delivered to and recovered from theeluate of each column in a bundle of columns as it passes through anobservation window machined into the exit capillary of that column witha fiber-optic cable.

Mating an Array of Through-Holes to an Array of Liquid ChromatographyChannels

A device can be manufactured to include an array of chromatographycolumns with a chromatography matrix immobilized within the array ofcolumns, for example, with spacing such that it can be co-registeredwith the through-holes in an array of through-holes that do not containa chromatography matrix. The physical size of the array of through-holescan determine the size of the array of columns. Preferably, the internaldiameter of each column in an array of columns will be similar to theinternal diameter of the corresponding co-registered through-hole in anarray of through-holes.

The array of columns should be mated to the array of through-holes in amanner that allows the application of a positive or negative pressureacross the device and does not result in cross talk between theindividual samples. The number of columns need not be in a one-to-oneration with the through holes. Radial diffusion of samples between thelayers of a stack of arrays of through-holes in response to an appliedexternal pressure may result in cross talk and must be avoided.Inter-sample cross talk can be eliminated by forming a liquid-tight sealhermetic seal between the through-holes to eliminate radial diffusion.An elastomer sheet with holes co-registered with the through-holes inthe array can be compressed between the layers of a stack to form such aseal. Alternatively, a thin, inert, porous polymer sheet can be placedbetween the two arrays such that, when the two columns are pressedtogether, liquid can flow through the pores in the sheet, but cannotflow laterally. Another approach entails manufacturing the array ofthrough-holes such that one side of the immediate area around eachthrough hole is raised relative to the rest of the array. An o-ring canthen be placed around this raised area. When two or more arrays ofthrough-holes are compressed together, the o-rings will form a hermeticseal, thereby eliminating radial diffusion and sample cross talk. Thecross-section of any pair of mating arrays can also be fabricated to beinterlocking with an elastomer gasket or coating between the matingsurfaces, to provide a leak-tight fluidic seal.

One or more arrays of through-holes can be mated to an array of columnsin a similar manner. A top plate can then be affixed to the assemblythat allows for the necessary chromatographic wash and/or elutionbuffers to be applied to the each through-hole. The liquid can be forcedthrough the device in such a manner that the sample will be pushedthrough the array of through-holes into the array of columns upon whichthe chromatography will take place. If required by a specificapplication, the wash and/or elution buffers from the chromatographycolumns can be transferred via capillaries to another chromatographydevice, chromatographic fraction collector, or another array ofthrough-holes. An illustration of such a device is shown in FIG. 14. Thefluidic connections that lead to and from the device can be machined foreasy coupling to standard fluidic connections using standard fluidiccomponents such as ferules and compression fittings.

Device for Fraction Collection

Chromatography generally entails separation of a mixture of compounds onthe basis of differential chemical and/or physical interactions of theindividual components of that mixture with a chromatographic matrix. Asudden or gradual change in the physical and/or chemical environment canaffect the interactions between the components of a mixture and thechromatographic matrix. Typically, each component of a mixture elutesindividually from the chromatographic matrix as the physical and/orchemical conditions are varied. It can be desirable to isolate a givencomponent of a mixture of compounds for further analysis orchromatography. In some cases, a component of interest can be identifiedby online spectroscopic analysis.

Devices for fractionating the eluant of a chromatographic column andstoring individual fractions are well known. One embodiment of thepresent invention features a system for collecting chromatographicfractions from an array of columns. The column eluate can be spotteddropwise onto another array of through-holes. By controlling the speedat which the array of through-holes is moved with respect to the arrayof columns, the volume of each fraction can be controlled. A fluidbridge can be formed between the exit capillary from the array ofcolumns and a through-hole in a through-holes array if the interiorsurface of the through-hole is coated with a material with theappropriate affinity for the eluant. The maximum number of fractionsthat can be collected from a given column will depend on the size ofeach fraction, on the speed at which the collection array ofthrough-holes is moved, and on the density of the array of columns. If alarge number of fractions must be collected from each column, a lineararray of columns can be used, its output being collected in atwo-dimensional array of through-holes. If the fraction collection arrayof through-holes is then moved perpendicularly to the linear array ofcolumns, the number of fractions that can be collected is limited onlyby the physical size of the collection array.

For some applications, a single chromatographic separation can takeseveral minutes or longer to complete. If long periods are required, itis possible that evaporation of liquid from the array of through-holesin the fraction collection device will occur. To avoid evaporative lossfrom the through-holes in the fraction collection device, the entirearray of through-holes used to collect fractions can be placed within anenvironmentally controlled enclosure. If a high-humidity environment ismaintained within the enclosure, evaporative losses can be minimized.Additionally, it can be desirable to maintain a certain temperaturewithin the enclosure (e.g., 4° C.) to maintain compound stability. Theelution capillaries from the array of columns can enter the enclosurethrough a series of precision-machined holes to maintain the integrityof the enclosure while allowing for introduction of the eluant from thearray of columns. Elastomer gaskets may be used to ensure a good sealaround the enclosure.

An array of through-holes containing the fractions collected from anarray of columns can be stacked with another array of through-holes toinitiate a second mixing operation to initiate a chemical reaction. Asecond chromatography application can be initiated by stacking the arrayof through-holes into which the fractions were collected with a secondarray of columns. Alternatively, further spectroscopic or spectrometricanalyses can be performed on the collected fractions at this time.

Spectrometric Analysis of Compounds in an Array of Through-Holes

Atmospheric Pressure Ionization Mass Spectrometry (API-MS)

Samples in an array of through-holes can be analyzed by a spectrometrictechnique such as atmospheric pressure ionization mass spectrometry(API-MS). The spectrometric analyses are typically performed serially.Therefore, the chips should be environmentally isolated in a controlledtemperature and humidity environment to avoid loss of sample due toevaporation. In API-MS, one simple method for introducing the sample tothe mass spectrometer features aspirating a selected sample directlyfrom a particular through-hole into a valve using a length of capillarytubing (e.g., as described herein). A metered volume of sample can thenbe introduced into a mass spectrometer using standard API-MS protocols.

Matrix Assisted Laser Desorption Ionization Time of Flight MassSpectrometry (MALDI TOF-MS)

In MALDI TOF-MS analysis, a sample of interest is generally mixed withone or more matrix-forming compounds. Typically, a saturated solution ofan organic matrix material (e.g., derivatives of hydroxycinnamic acid)is mixed with an equal volume of sample. In some applications of MALDITOF-MS, the organic matrix compound is replaced by inorganicnanoparticles (e.g., colloidal gold, quantum dots, or porous silica).The mixture is then spotted in the form of a regular and addressablearray on a flat plate and allowed to evaporate completely. The sampleplate is then positioned in the mass spectrometer, and the samples areionized by irradiation from a pulsed laser.

Samples in an array of through-holes are well suited for analysis usingMALDI TOF-MS and related applications, since the necessary samplepreparations steps can easily be accomplished in a parallel fashion. Forexample, a second array of through-holes can be loaded with a saturatedsolution of an organic matrix or a slurry of an inorganic matrixcompound. The array of through-holes can either be dip-loaded uniformly,or, if desired, any number of different matrix compounds can be loadedinto individual through-holes in an addressable fashion. The sample andmatrix arrays of through-holes can be mixed together by bringing thechips together (e.g., as described herein). After allowing the solventto completely evaporate, the array of through-holes can be placed in aslightly modified receptacle in most commercially available MALDI TOFmass spectrometers. The conventional flat metal MALDI plate can bemachined down to compensate for the thickness of the array ofthrough-holes. The array of through-holes can be affixed with atemporary adhesive within the recessed area of the standard sampleholder.

The laser used for sample ionization in the MALDI TOF mass spectrometercan be focused within the through-hole to provide the necessaryirradiance for sample ionization. Internal reflection of the laser beamwithin the through-hole can possibly increase the amount of laser energyabsorbed by the matrix and transferred to the sample, thereby increasingthe amount of sample ionization. Additionally, an array of through-holescan allow for a very high density of samples to be spatially located ina small footprint without inter-sample contamination.

Typical MALDI-MS sample plates are solid surfaces onto which samples arespotted. The laser used to ionize the samples must be on the same sideof the plate as the inlet of the flight tube of the mass spectrometer,since the sample plate is opaque to the laser energy. The use of anarray of through-holes as the sample plate allows for the source oflaser irradiation and the inlet to the TOF mass spectrometer to belocated on opposite faces of the sample plate. A scheme of this linearMALDI TOF mass spectrometer is shown in FIG. 15. Translocation of thesample plate in front of the inlet of the flight tube allows for thelaser ionization of a selected sample.

Alternatively, an array of posts or pins, precision-machined to fit intoan array of through-holes, can be coated with the MALDI matrix materialby dipping the array into a bulk matrix solution. After the solvent hasevaporated, the pin array can be inserted into the through-hole array.Fluid contained in each through-hole is transferred to the correspondingpin surface. After the solvent has evaporated, the pin array can beplaced at the input to a TOF mass spectrometer and the pins can beilluminated sequentially with a focused laser beam. In such a pin array,a portion of the sample from each through-hole can be held isolated fromits neighbor by the air gap between each pin.

Through-Hole Array/Surface Method.

When placed into an electric field or driven by pressure, thethrough-hole array can be used as a parallel capillary electrophoresis,electrokinetic chromatography or chromatography device. An array ofsamples in one through-hole array can be introduced into a second,typically longer, through-hole array. The second through-hole array canbe filled with a gel (e.g. silica), a polymer (e.g., polyacrylamide) ora resin and can have a coating on its walls to prevent or enhanceelectro-osmosis or protein binding. However, if electrophoresis orchromatography is performed in such an array, it will be difficult toanalyze the output of each column as molecules emerge from it. One wayto alleviate this problem is to pass the output through a moving surface(e.g, nitrocellulose sheet) with an affinity for the analyte moleculesand then move the web to an imaging detector. For example, fluorescentlylabeled DNA or protein could be eluted onto a moving nitrocellulosemembrane and passed to a fluorescent imager to analyze. A continuouslymoving surface, moving in the manner of a tape, would cause smearing ofthe samples, therefore it is advantageous to reduce or reverse thepolarity of the electric field or pressure during those periods of timewhen the surface is moving. An increase in sensitivity of the detectionsystem is can be achieved by further delaying the movement of thesurface and the voltage or pressure while the detector is acquiring animage. Alternately, the imager could be a line-scanner such as afluorescence laser line-scanner. The surface could be fed from a longspool if desired (e.g., in a tape like manner). The surface can furtherbe taken up on a second spool.

Readout Methods. A Method of Using Wetting Properties of the Array toDetect Chemical Binding to the Walls of the Through-Holes.

The binding between two proteins, such as between an antibody and anantigen is detected via its affect on the surface energy of the channelinteriors.

In a preferred embodiment, a library (e.g., a library of antibodies,receptors, macromolecules, or molecular probes) is bound to the interiorwalls of a through-hole array having hydrophilic channel walls andhydrophobic faces. The array is rinsed with a blocking agent, whichbinds to non-specific protein absorption sites on the channel walls butdoes not change the hydrophilic character of the wall surface. Suchblocking agents include bovine serum albumin (BSA), powdered milk, andgelatin. The array is then immersed in a solution that contains antigenand is incubated for sufficient time to allow binding of antigen tocomplementary antibody. The arrays are then removed from the antigensolution and washed with buffer. The array is dried and dipped into anaqueous solution containing a chromophore or fluorophore. The presenceof the antigen on the surfaces lowers the surface energy sufficientlysuch that the liquid is prevented from entering the through-holes. Theempty through-holes can be identified by imaging the array. The emptythrough-holes then correspond to the antibodies in the library thateffectively bind the ligand.

In another embodiment, the interior walls of a set of small arraydevices, each having roughly 1-100 through-holes, are coated such thateach array has a different antibody. The arrays are placed together in asolution of antigen and incubated for sufficient time to allow bindingof antigen to complementary antibody. The arrays are then removed fromthe antigen solution and washed with buffer. All arrays are then placedtogether in a separation bath that contains a liquid that isnon-destructive to the proteins and can be adjusted in density in somemanner (e.g., by addition of a higher density liquid, or by adding athickening agent).

When the holes remain empty, the density of the array in the bath isreduced and, under appropriate conditions, the array containing boundantigen can float to the surface. The presence of the antigen on thesurfaces lowers the surface energy sufficiently such that the liquid isprevented from entering the through-holes. These floating arrays areremoved and the identity of the antibody bound to each array isdetermined by mass spectroscopy, or from a code on each array, or bysome other means such as a bar code or radio transponder built into thearrays. For this method the array can be a porous structure such as anaerogel or a porous bead.

A Device for the Analysis of an Array of Through-Holes by MassSpectrometry.

Samples or aliquots of samples can be removed from an array ofthrough-holes for analysis by mass spectrometry by one of severaldifferent methods. One such method features drawing the sample or analiquot thereof into a tube with the application of negative pressure.In one example of this approach, the tip of a syringe is inserted into aselected through-hole in an array and a metered amount of sample isdrawn into the syringe. Alternatively a vacuum could be used to aspiratethe samples into a length of tubing, a valve, or a container forstorage.

For certain applications, it can be desirable to assay each sample in anarray of through-holes by a serial process such as mass spectrometry.Application of a serial process to a large number of samples in an arrayof through-holes, even if done very rapidly, can still require asignificant amount of time. If humidity conditions and temperature arenot strictly regulated during this time, evaporation of samples from thethrough-holes can occur and artificially bias assay results.

One approach to controlling evaporation and facilitating the aspiratingof individual samples from an array of through-holes is to design anadditional array of through-holes in which each of the through-holes iscoregistered with a sample through-hole in the assay. This additionalarray of through-holes can be placed on top of the arrays ofthrough-holes used in the assay to create a top plate. The through-holesin this top plate can be designed such that the diameter of eachthrough-hole can be made to be much larger at the outer surface than thediameter in the surface that contacts the arrays of through-holes usedin the assay. The conical shape formed by such a through-hole will actas a guide for a syringe needle into a selected through-hole andfacilitate efficient sampling. The outer surface of this top plate canalso be coated with a thin film of polymer similar to that used inlamination. The sealed surface will act to retard evaporation. Thesyringe needle used for aspirating the sample out of the through-holescan easily perforate this thin film and will not hinder efficientsampling.

Once the sample is aspirated into a syringe, it can be delivered into amass spectrometer for analysis by any one of many techniques known bythose skilled in the art. These can include atmospheric pressureionization techniques such as electrospray ionization (ESI) oratmospheric pressure chemical ionization (APCI).

In another embodiment of the invention, a metal plate can be used as abottom plate for the array of through-holes. The solvent used in theassay can be allowed to evaporate and a solid sample can form in afootprint on the bottom plate that corresponds to the internal diameterof the through-hole. If desired, a matrix can be added to the samplesbefore complete evaporation. Alternatively, a matrix can be added to thesurface of the metal bottom plate before it is stacked with the arraysof through-holes used in the assay. Once the samples have completelyevaporated the metal bottom plate can be removed from the arrays ofthrough-holes and each sample can be analyzed by matrix assistedlaser-desorption ionization (MALDI) or a similar surface basedionization mass spectrometry technique generally practiced by thoseskilled in the art.

In another embodiment of the invention, an array of pins coregisteredwith the array of through-holes can be dipped into the array ofthrough-holes and removed. Sample that is residually removed with thearray of through-holes can be allowed to evaporate on the tips of thearray of pins. As in the previous embodiment, this evaporated sample canbe used for a surface based mass spectrometry method.

Time-Gated Fluorescence Imaging of a Through-Hole Array

Many biological assays are configured to give a fluorescent readout thatcan be acquired from an array of through-holes by fluorescence imaging.Typically, light from an excitation lamp or laser is passed through anexcitation filter, through the array, through an emission filter andthen to a CCD camera. In many cases, the sensitivity of the signal islimited by background light due to imperfect performance of the filters,and by inelastic and elastic scattering of light by the sample andoptical components. Whereas the fluorophores of interest havefluorescence lifetimes of about 1 ns to 1 ms, scattering occurs at muchshorter timescales. Thus removal of background light can be accomplishedby the technique of time-gating. Time-gating the process of illuminatingthe sample while preventing the camera from acquiring data, quicklyremoving the excitation light, then waiting for a delay time beforeacquiring the fluorescence emission image. By not collecting photonsemitted during the first 1 to 100 ps of after excitation, backgroundnoise is significantly reduced and signal to noise is improved. Asimilar apparatus can be used to repeat the data acquisition withvarying delay times, thus yielding fluorescence lifetime information foreach of the through-holes in the array.

Various strategies can be used to construct a time-gated fluorescenceimaging system. A pulsed excitation source is needed and can be either aflash lamp or laser such as a passive or active mode-locked orQ-switched laser. If a laser is used, a beam expander and diffuser platewill give uniform irradiation of the platen. A continuous excitationsource can also be used with a means for rapidly blocking andun-blocking the light such as an electro-optical, an acousto-opticalcell or a rapidly rotating disk with slits. A pulse generator can beused to trigger the illumination source and detector at a given delay.The CCD camera can be electronically shuttered or physically shutteredas with a rotating disk with slits that is out of phase with theexcitation pulsing.

Optical Readout Based on Insertion of a Through-Hole Array into anOptical Resonator or Interferometer

A through-hole array can be inserted into either an optical resonator oran optical interferometer for simultaneous and parallel interaction ofan optical field with material contained in each through-hole. (See FIG.21) Such a method can be advantageous when the through-hole array (2) isplaced in an optical resonator (Irradiation (1) is shined and amplifiedbetween mirrors (2) and (3).) so that the optical path length isincreased over the length of the through-hole array, to increaseabsorption (5). The optical path length is increased as a multiple ofthe through-length, to increase optical absorption. For example, forsimultaneous initiation of chemical reactions by the enhanced opticalfield characteristic of an optical resonator. It can also beadvantageous as a means for simultaneous analysis of materials andinteractions between materials contained within the through-holes, forexample, by recording changes in incident optical field intensity,phase, polarization, or frequency, or by recording of these parameters,of light emitted from as a result of interaction between an incidentoptical field (e.g. fluorescence, phosphorescence), the materialscontained in a through-hole, or a change in the material itself (e.g.luminescence).

One advantage of measuring these parameters with the chip as part of anoptical resonator is that the resonator's resonance condition willchange on interaction of the optical field contained within theresonator structure with the materials contained within eachthrough-hole. These changes (e.g., phase, intensity, polarization,frequency) are intimately related to the composition and physical stateof the material contained in the through-hole. The change in opticalfield parameters changes the resonant condition of the cavity, which, inturn, changes the intensity of light passed through or reflected fromthe resonator.

One can also take advantage of the increased optical field strengthcharacteristic of optical resonant structures, for example, to initiatephotochemical reactions or non-linear optical effects (multi photonabsorption, harmonic conversion, etc.) as a probe to measure propertiesof the materials contained in the through-holes. The optical fieldincident on the resonator can be either continuous in time or it canvary with time as in an optical pulse.

Examples of optical resonator structures that can be used in thisembodiment include Fabry-Perot-style interferometers with two planarmirrors and confocal Fabry-Perot-style interferometers having two curvedmirrors or one planar and one curved minor.

The through-hole array can either be part of or be inserted into atwo-beam interferometer. Examples of such interferometers are many, andinclude Michelson, Twyman-Green, Sagnac, and Mach-Zhender-typeinterferometers. In the embodiment where the array is inserted into oneof the optical paths of a two-beam interferometer, the phase of thelight is delayed according to the complex refractive index (refractiveindex and absorption) as a function of wavelength. The interferometercan be illuminated with a beam of white light of sufficient width toalso illuminate the through-hole array. For each optical path lengthdifference between the two arms of the interferometer, a camera at theinterferometer output can record the pattern of light exiting theinterferometer and corresponding to light that has passed through eachhole in the array. A series of images can thus be acquired for eachoptical path length difference, and taking the Fourier transform of eachpixel of the image as a function of optical path length can generate anoptical absorption or emission spectrum for the materials in eachthrough-hole of the array.

Another embodiment uses a two-beam interferometer to analyze lightpassed through or emitted from a through-hole array. A camera recordsthe light pattern from the interferometer for each optical path lengthdifference imposed between the two plane minors that make up theinterferometer. After recording a sequence of images corresponding toeach path length difference, individual interferograms can be generatedfrom each set of pixels co-registered across the image sequence.Application of the Fourier transform to each interferogram can generatean absorption or emission spectrum at each spatial position in theimage. In this way, the spectral content of light interacting withmaterial contained in each through-hole of the array can be determined.

Application of the approach described in U.S. Pat. No. 6,088,100,incorporated herein by reference in its entirety, adapted for full-fieldimaging, can also allow for capture of absorption spectroscopicinformation from each through-hole in a stack of through-hole arrays.

Image Center of Array as a Function of Thermal Perturbation.

The invention is compatible with many systems for detecting the outputof the arrays of chemical probes. Commercially available fluorescencescanners can be used if desired. Because each position in the array hastwo apertures (i.e., on the top and bottom faces of the platens), thearray can be exposed to electromagnetic radiation on one face of theplaten, and the optical properties of the samples in the array can bemeasured via detection at the opposite face of the platen. The positionsof the array can be imaged in parallel or by serial scanning techniques.Both static and kinetic analysis of reactions can be utilized.

One method of detecting binding between an analyte and probesimmobilized in the walls of the through-holes includes observing thedistribution of analyte within each through-hole as a function of aperturbation. For example, a ligand of interest can be covalentlyattached to the inner walls of each of 10,000 through-holes in a 2 sqcm. platen. The chip can then be stacked with another chip containing,for example, a peptide library such that each member of the libraryoccupies its own through-hole and has a fluorescent tag. After allowingsufficient time for non-covalent binding reactions to reach equilibrium,the chip can be rinsed with buffer to remove unbound material. Byobserving the fluorescence distribution in each hole as a function of aperturbation such as increasing temperature or increasing formamideconcentration, the members of the library can be ranked as to bindingenergy. Binding kinetics can be determined by following the fluorescencedistribution as a function of time following a rapid perturbation suchas a temperature jump. The sensitivity of the assay can be improved byusing a mask that includes of an array of through-holes complementary tothe chip, but of smaller diameter, that spatially filters the light suchthat only the interior of each through-hole is observed. Another methodof increasing signal-to-noise ratio includes applying a periodic orstochastic perturbation such as temperature, and then observing onlysignal correlated with the perturbation.

An increasingly common technique in drug discovery is millisecondtime-scale fluorescence analysis of cell populations. Existingcommercially available devices utilize a bank of syringes that addreagents from one 384-well microplate containing drug candidates to asecond 384-well microplate containing cells loaded with a fluorescentindicator of calcium such as Fura-2, followed by laser scanning of theunderside of the second plate to generate millisecond kineticmeasurements of calcium release from the endoplasmic reticulum of thecells. It would be advantageous to use a white-light excitation sourceand a digital camera to acquire such kinetic data due to lower cost andgreater choice of excitation wavelengths. This was previously difficultbecause the syringe bank would obstruct the light path in such a systemand would hayed to be moved on a millisecond time scale, which is nottypically possible. Use of a white-light source is possible byinitiating mixing of samples stored in an array of through-holes withcells growing in a second array, both arrays being in the camera system.An additional benefit of this method is that the throughput of samplescollected is greatly increased by the use of through-hole arrayscontaining as many as 20,000 or more samples. Typically, the system isautomated and will collect data that begins at the moment of mixing orotherwise indexes the times associated with collected data points to themoment of stacking. The types of assays possible with this method arenot limited to cell or fluorescence assays, any assay with an opticalread-out that occurs on a time scale of less than minutes can benefitfrom the invention.

A preferred embodiment of the invention includes at least two stackedand co-aligned platens containing through-hole arrays consists of (i) adetection device; (ii) a means for introducing platens into thedetection device (iii) a means to register the platens to cause fluid tocommunicate between at least some of the co-registered through-holes and(iv) a means of contacting the platens to initiate mixing of reagentssimultaneously in at least some of the through-holes. The detectiondevice (FIG. 24) can be an imaging device, such as a CCD camera, withoptical filters and a light source for illumination. Light from anoptical source (1) illuminates parabolic collimation mirrors (3) afterpassage through a flexible, bifurcated fiber bundle (2). The light thenilluminates the stack of arrays at an oblique angle such that light raystransmitted through the array through-holes does not enter into thecamera lens (5). This optical arrangement is desirable as a simple meansto decrease optical background for increased optical sensitivity.

Separation Methods.

A Method of Separating a Stack of Two or More Arrays Filled with Liquid.

It can be desirable to separate arrays once the contents of individualthrough-holes have been combined by stacking. For example, in order toperform a dilution by two an array filled with a chemical library isstacked onto an array containing solvent buffer. After sufficient timefor mixing of the two sets of liquids (approximately 15 seconds for 100n1), the plates are separated to produce two identical arrays oflibraries members at half the initial concentration. This process can berepeated to produce a dilution series.

When two through-hole arrays are stacked such that their contents mix,the two plates are not readily separated with out cross-contaminationbetween neighboring through-holes. Pulling one plate against theadditive surface tension created by many microscopic columns of fluidinvariably introduces shear forces that mixes the contents of individualthrough-holes together as the chips are separated.

A method is proposed to separate each fluid column into two shortercolumns separated by a small vapor phase. Small electrodes at theinterface between the two stacked arrays produce a small volume of gasin each through-hole. As the bubble grows it recreates the liquid vaporinterfaces between the two arrays. After each column is cleaved in twothe arrays are separated mechanically.

Alternatively an inert, humid gas is introduced into the atmosphereabove and/or below the stacked arrays and nucleated at the interfacebetween the two stacked arrays.

Alternatively the gas can be pumped into the center of each through amatching array of very fine hollow tubes.

Centrifuging (Performing Gravimetric Separation in) an Array ofThrough-Holes.

Many biological or chemical assays require centrifugation and/orfiltration of samples. In many it can be desirable to filter orcentrifuge samples in a biological or chemical assay that is performedin array of through-holes. The following invention pertains to a devicefor the centrifugation and or filtration of an array of through-holes.

A metal jig with two flat surfaces larger than the array ofthrough-holes can be built. A single or a stack of arrays ofthrough-holes are placed between the two flat surfaces and evenlycompressed together with the application of force on the metal plates.The metal jig will be machined such that the amount of compression canbe adjusted as desired, preferably by a simple tightening of severalscrews holding the jig together.

In some applications the filtrate or pellet will need to be recoveredfrom the centrifuged sample. In other cases the pellet formed aftercentrifugation will need to be removed. A simple solution for this is tomachine a plate that contains an array of wells or dimples of a meteredvolume that are coregistered to the array of through-holes. The array(s)of through-holes can be stacked atop this bottom plate with coregisteredwells. If desired, a filter can be placed between the bottom plate andthe array(s) of through-holes. After the centrifugation is complete, thebottom plate can be removed and the filtrate or contaminatingprecipitate can be removed. Alternatively, if the supematant is thedesired fraction, it can be aspirated directly from the array ofthrough-holes without the need for removal of the bottom plate.

In certain applications a large centrifugal force can need to be appliedto the array(s) of through-holes. Even with a bottom plate and/or afilter application of large amounts of centrifugal force a stack ofarrays of through-holes can result in a lateral displacement of samplesthat are forced into the spaces between the individual layers of thearrays of through-holes. Coating of the contacting surfaces of the arrayof through-holes with a hydrophobic material will inhibit lateraldiffusion between layers at relatively low centrifugal forces. When highlevels of centrifugal forces are required a material can be stackedwithin each individual array of through-holes such that when the arrayof through-holes are compressed together by the metal jig a leak-tightseal will be formed between the two arrays of through-holes. If thematerial used to form the seal is porous it will not impede the flow ofliquid between the layers of through-holes, yet it will form a tightseal against lateral flow.

VI. Miscellaneous Uses.

To fully realize the potential of reagent volume savings and increasedthroughput provided by nanoliter volume fluid handling, means andmethods of storing chemical and biological samples at high density inlow volumes are necessary. These storage systems must be easily accessby microfluidic screening instrumentation.

One embodiment of the invention provides the a method to store chemicalor biochemical samples at high density and in low volumes. Additionally,samples stored using the devices and methods of the invention can beassayed with a minimum of liquid handling steps or other manipulations.The method includes placing a small volume of compounds dissolved in asolvent in an array of through-holes and adding a second solvent to thearray of through-holes without causing substantial migration of thecompounds out of the through-holes. The result is an array of compoundsdissolved in a liquid that is primarily comprised of the second solvent.

The invention further provides a method of storing compounds in a mannerwherein the samples can be readily introduced into aqueous medium forperformance of an assay. The step include dispensing a volume ofcompound in a solvent that is much less than the volume of the containerthat it is dispensed in, storing for some time, and adding an aqueousmedium to fill the remaining volume of the container in preparation foran assay.

In a preferred embodiment, integration of low-volume compound librarystorage and screening includes the following steps: (1) Dispensingvolumes of compound dissolved in a non-aqueous solvent that are smallerthan the total volume of the containers to be used for screening.Usually, the volume dispensed will be less than half and could be 1/10thof, 1/40th of, or less than the total capacity of the container.Usually, the containers will be part of an array of containers. Thecontainer is preferably a hydrophilic area surrounded by hydrophobicareas, such as a channel of a through-hole array, or a spot on a glassslide with spots of hydrophobic areas in a hydrophilic background. (2)Storing the compounds for some period of time. Storage conditions mayconsist of a low temperature such as 4° C., −20° C., −80° C., submergedin liquid nitrogen, or a lower temperature. Desiccation is usuallydesirable. (3) Removing the compounds from storage, and elevating themto above the freezing point of the aqueous media to be loaded into thecontainers, but above the freezing. (4) Adding aqueous medium to thecontainers. Preferably, the aqueous medium is chilled to above itsfreezing point and below the freezing point of the non-aqueous medium.The addition could be done by dispensers, but is more rapidly andinexpensively done by dipping the containers into a bath of the aqueousmedium. It is advantageous to have the non-aqueous solvent be in thesolid form so as to minimize loss of the compounds into the aqueousmedium and to prevent communication of compounds in adjacent or nearbychannels. (5) Waiting for a time sufficient to allow mixing of thecompound with the aqueous medium. And (6) Performing an assay ormeasurement upon the samples.

Methods for Dispensing Small Volumes into Large Holes.

It is advantageous to screen samples such as drug candidates in verysmall volumes in order to conserve reagents and increase through-put. Atypical through-hole array format will have channels that hold 60 n1 offluid and a typical assay will be done with two stacked chips, holding atotal of 120 nl. If the maximum concentration of DMSO acceptable in theassay is 2%, then a total of 2.4 nl must be dispensed into one of thechannels and this is extremely difficult to do with conventional liquidhandling systems. One way to solve this problem is to dissolve thecompounds in a volatile solvent such as ethanol, DMSO, water, dispensethe compounds and allow the solvent to evaporate, leaving a spot ofdried compound in the container. This has some major disadvantages inthat the compound may crystallize into a form that does not easilyre-solubilize, and is not sufficiently immobilized. Another approachwould be to dispense the compound in a volatile solvent such as DMSO andallow the DMSO to evaporate to leave the desired compound in a lowervolume of DMSO. In this case, it may be difficult to evenly evaporatethe solvent, which may interfere with the assay to be performed on thesamples.

A more robust approach is to dissolve the compound in a first volatilesolvent such as DMSO and a second, more volatile solvent, such asethanol or methanol, dispense the mixture into the containers and allowthe second solvent to evaporate, leaving compound dissolved in to firstsolvent. Ethanol and methanol are good choices since they will dissolvemost drug-like compounds and are hydrophilic enough to remain containedin a hydrophilic container surrounded by hydrophobic barriers, althoughother solvents could be used. DMSO is a good solvent for use as thefirst solvent since it dissolves most compounds, will stay in place dueto its hydrophilicity and is not very volatile, evaporating more slowlythan water.

The invention also features a method for storing compounds dry in anarray of sub-microliter containers separated by hydrophobic barrier.Compounds are dissolved in a volatile solvent or combination ofsolvents, introduced into the array of containers and the solvent isallowed to dry, leaving a thin film of solid compound. If necessary, abiocompatible adhesive is added to keep the film attached to the wallsof the container. A solvent mixture containing a first volatile solventand a second less-volatile solvent is added to substantially all of thecontainers in the array. In a preferred embodiment, the solvent mixturecomprises an alcohol such as ethanol and DMSO. The more volatile solventis allowed to evaporate, leaving a residue of the less-volatile solventthat dissolves at least most of the compounds in the array. Typically,the less-volatile solvent will comprise the minority of the solventmixture, is usually less than 20%, and is often less than 5% of thesolvent mixture. The array is then prepared for assay by introducing anaqueous media that is compatible with the assay such as water or abuffer. The aqueous media may be dispensed by the methods describedabove.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 DNA Probe Array

A 50,000-channel through-hole array is fabricated from silicon. Usingthe gene database, series of 80 spatial filter top masks with another 80identical bottom masks are fabricated. Arrays are aligned andderivatized with 3-glycidoxypropyltrimethoxysilane in order to provide afree functional group coating on the interior surfaces of thethrough-holes.

A stack of ten coated platens is aligned. Alignment is verified byobserving the optical transmission of the various channels. Mask #1 isaligned with the top of the stack and an identical mask aligned in thesame orientation and the bottom of the stack. Mask #1 is constructedsuch that the positions in the mask that corresponded to a gene in thedatabase with an adenosine in the first position are open and allow flowthrough the through-holes in those addressable positions. The stack isrinsed with dry acetonitrile. A phosphoramidite monomer at aconcentration of 0.1 M acetonitrile and tetrazole is added by apressurized flow against the mask. The coupling reaction is allowed toproceed for 3 minutes. An oxidizing solution ofiodine/lutidine/acetonitrile/water is introduced into the chip stack andincubated for 2 minutes, followed by rinses with acetonitrile anddichloromethane. The chip is dried by vacuum.

The masks are removed and replaced with a top and bottom Mask #2corresponding to positions to which a T monomer is to be added. Adeprotection agent is added, and the T coupling reaction is commenced.

This process is repeated with Mask #3 for G in the first position, andMask #4 for C in the first position. Mask #5 corresponds to A in thesecond position, and so on. After the synthesis is complete the chip isstored in 30% ammonia for 12 hours.

To test the array, a fluorescein end-labeled 20-mer corresponding to oneof the intended probes in the array is synthesized by standard methods,dissolved in a hybridizing solution of 6×SSC/0.5% SDS, and introducedinto all through-holes of the array simultaneously. The array is washedwith 0.5×SSC and the chip is imaged fluorescently. Only the position inthe array corresponding to the probe complementary to the testoligonucleotide shows a significant increase in fluorescent intensityover the background level determined by averaging the signal from theremaining positions in the array.

Example 2 Catalyst Screening

A recombinant enzyme library is screened in a dense array ofthrough-holes against a fluorogenic substrate. Genetically diverse E.coli containing the gene for the protease subtilisin with a polyhistidine tag is created by mutagenesis. A dilute solution of thebacteria is added to a nickel-coated array such that there is an averageof 1 to 2 bacteria per through-hole. The bacteria are allowed to grow tothe log phase and replicate plated (as described above). The bacteria inthe array are lysed by heating, allowing the tagged subtilisin to attachto the nickel coated walls of the array. Another array containing thefluorogenic substrate and reaction buffer (Boehringer) in each well isstacked with the first array. The stack is immediately placed in a CCDcamera-based fluorescent imaging system, and the rate of increase influorescence intensity is measured for each through-hole. The enzymewith the fastest rate is selected and the corresponding bacteria in thereplica plate are grown for further studies.

Example 3 High Throughput Screening with Beads

A 100,000 member combinatorial library immobilized via a photocleavablelinker on 10 micron diameter beads is purchased from Affymax. A platenis prepared with 100,000 through-holes of a diameter such that only onebead fits in each hole. The beads are washed in PBS, suspended in asolution containing a fluorogenic substrate for the enzyme to bescreened, and spread over the platen with a rubber spatula. Anultraviolet lamp is used to decouple the members of the library from thebeads. A second platen filled with a solution of the enzyme in areaction buffer is aligned and stacked on top of the first chip. Thestack is immediately imaged by epi-fluorescence to determine the rate ofincrease in fluorescence intensity as a function of channel position.Those holes that exhibit decreased enzyme rates are selected for furtheranalysis as drug leads.

Example 4 Absorption Assay

A single-cell layer of CaCo-2 cells is grown on two identicalanisotropic membranes coated with collagen slightly larger than thearray of through-holes. The cell culture conditions, media, and membranecoatings used in the in vitro growth and maintenance of CaCo-2 cells arewell known by those skilled in the art. Once a uniform layer of cells isestablished, each membrane is sandwiched between two identical arrays ofthrough holes dip-loaded with culture maintenance media. The array andmembrane assemblies are cultured an additional day to allow for theCaCo-2 cells to equilibrate and form an intact layer within thethrough-holes. Two additional arrays with through-holes co-registeredwith the CaCo-2 arrays are loaded with a chemical diversity library ofsmall molecules. A compound known not to pass through CaCo-2 cells (e.g.mannitol) is used as a negative control to assess the integrity of theCaCo-2 cell monolayer, while a compound known to easily diffuse throughCaCo-2 cell monolayers is used as a positive control. One of the arrayscontaining the chemical library is stacked on the apical side of one ofthe CaCo-2 cell monolayer arrays to assess apical to basal absorptionwhile the second identical chemical library array is placed on the basalside of the other CaCo-2 cell monolayer array to assess basal to apicalabsorption. The completed arrays are incubated for 1 hour, allowing timefor transport or permeation of the library compounds. The oralbio-availability of the chemical library is assessed by quantifying theamount of library compound diffused through the CaCo-2 cell monolayer(e.g. by mass spectrometry).

Example 5 Ligand Fishing by Blotting from a 2-D Gel

Cellular proteins exhibiting an affinity for a ligand are identifiedusing a 2-D gel. A platen having 500,000 through-holes is derivatized inorder to covalently link a segment of the human epidermal growth factorreceptor to the inside of each through-hole. Each hole is filled with abuffer solution. A cellular extract of a human cell line is thenseparated on a 2-D gel of a size similar to that of the platen, and thenaligned with the platen. The proteins in the 2-D gel are then blottedonto the chip by applying a buffer above the chip and drawing fluidthrough the chip by placing the chip on a blotter. The proteins are thustransferred through the chip and those that have an affinity for theepidermal growth factor receptor are retained in the chip. A denaturingsolution composed of 1M formamide in 100 mM Tris buffer at pH 8 is usedto blot the contents of the chip onto a platen for mass spectrometricanalysis. The location of those holes that contain a protein withaffinity for the receptor and the mass of the proteins contained inthose holes are used to determine the identity of the binding proteins.These proteins can be targets for drugs that block the EGF signalingpathway as a treatment for certain cancers.

Example 6 Screening for Antibiotics

A 500,000 member combinatorial peptide library is prepared in a platenthat includes 500,000 through-holes such that the peptides are dissolvedin a sterile cell culture medium within the holes. The library isprepared such that the identity of the peptide present in eachthrough-hole is known. A dilute culture of Enterococcus faecium bacteriais prepared such that each through-hole will receive on average 10bacteria in cell culture medium. The bacteria platen is stacked with thepeptide library platen to achieve mixing then incubated at 30° C. for 5hours. The stacked platens are then imaged by light scatteringmeasurement to determine the degree of bacterial growth in eachthrough-hole.

The holes showing a greater than 99% reduction in growth are identified,and larger quantities of the corresponding peptides are synthesized forfurther analysis.

Example 7 Finding the Peptide Target of a Kinase by Radiolabeling

A protein suspected of being a protein kinase is isolated. The proteinis incubated with radiolabeled ATP substrate in the presence of 100,000different proteins, all occupying unique positions in a platen havingthrough-holes. After incubation for a sufficient time (e.g., about 20minutes), the platen containing through-holes is washed with water andthe presence of radiolabeled proteins is detected by a phosphor-imagingsystem. The protein target for the kinase is thus identified.

Example 8 Cytochrome P450 Inhibition Assays by Fluorescence

The purpose of this experiment is to examine the potential of a libraryof compounds to inhibit a specific CYP450 enzyme. The protocol isadapted from Crespi et al., Anal. Biochem. 248:188-190, 1997. Thefluorometric substrate is 3-cyano-7-ethoxycoumarin for CYP1A2, CYP2C9,CYP2C19 and CYP2D6, and resoufin benzyl ether (BzRes) for CYP3A4. Thesereagents are obtained from Pharmazyme. A compound library is loaded intoone platen array device for each of the CYP450 enzymes to be tested at aconcentration equivalent to the Km of each enzyme. The appropriatesubstrate containing reaction mixture is added to a second platen arraydevice and the enzyme is added to a third platen array device. The chipsare stacked to initiate the reaction, and the increase in fluorescentsignal is monitored continuously by fluorescent imaging. The relativerates of P450 inhibition are used to select a drug lead from thecandidate compound library.

Example 9 High Throughput Protein Crystallization

A protein in a solvent is uniformly loaded into a through-hole array.The array through-holes are randomly filled with solutions containingdifferent salts, randomly changing the concentration and relativeabundance of the salts. Acidic and basic solutions are subsequentlyloaded into the through-holes, varying the pH. A temperature gradient isapplied to the array device (or the array can alternatively be held at aconstant temperature), causing the solvent to evaporate at a given rate.Additionally, the partial pressure of solvent can also be changed in thecontainer in which the array is placed to change the evaporation rate.Those through-holes in which protein crystallization is observed areexposed to a beam of X-rays or electrons and the diffraction patternrecorded for analysis. An important benefit is the rapid and efficientdiscovery of experimental conditions leading to crystallization.Furthermore, protein crystals can be analyzed directly in thethrough-hole array.

Sixteen different crystallant solutions, and eleven different buffers,are obtained from Emerald Biostructures (Bainbridge Island, WA), andrandomly loaded into the platen through-holes together with lysozyme. Atemperature gradient is applied across the platen, and the excess liquidis removed with a rubber spatula. The system is sealed in a containerwith 20 ml of precipitant solution. Optimal solution conditions aredetermined for crystallization using LC-MS (liquid chromatography-massspectrometry).

Example 10 Ultra High Throughput Mixture Separation and Screening inDense Arrays of Through-Holes

In many situations such as screening of natural products forpharmacologically active molecules, complex mixtures need to be rapidlyand efficiently separated and screened against protein targets. Thepurpose of this experiment is to separate and screen in a dense array ofthrough-holes a complex mixture of natural products against afluorogenic substrate. The natural product sample is first prepared inthe normal manner for high pressure liquid chromatography (HPLC). Asliquid elutes from the chromatographic column, equi-volume samples areacquired and stored sequentially in the array through-holes. A replicateplate can be generated simultaneously. Fluorogenic substrate is thenloaded uniformly into a second through-hole array. After completion ofchromatographic separation, each sample in the array is exposed to thesubstrate by stacking the sample plate onto the substrate plate. Opticalmonitoring the fluorescence signal from the assayed fractions selectssamples for further evaluation either from the assayed plate or thereplicate plate.

An extension of this method to applications where multiple mixtures arestored in a dense array of through-holes is also described (e.g.,separation and identification of the active component in a mixture). Inone embodiment, a capillary tube array having the same center-to-centerspacing as through-holes in the platen is brought into contact with thesample array. Each tube is located to spatially address one hole in thearray. The opposite end of the tubing array is inserted into a stack ofarrays where each capillary tube addresses a single column ofthrough-holes on spatially co-registered arrays. The number of stackedarrays equals the number of elution samples to be captured and analyzed.Each tube is pre-filled with a porous gel suitable for chromatographicseparation of the mixtures. An array plate filled with buffer is stackedonto the sample plate and a pressure is applied to drive the bufferthrough each through-hole and into the corresponding capillary tube. Asliquid exits from each tube, the through-hole in which the tube residesis filled. As the tube array is slowly withdrawn from the array stackthe liquid sample is retained in the through-hole. One advantage of thisscheme is that fractions eluted from the array of capillary tubes aresimultaneously collected. Once an array is filled, it can be removedfrom the stack and assayed, (e.g., contacting with a platen arraycontaining a fluorogenic substrate). Active compounds revealed byfluorescence emission are then removed from the plate for analysis.Alternatively, the flow rate of liquid from the tubing array andwithdrawal velocity can be chosen such that two plates are filled withessentially the same fraction eluted from the column. The two plates areremoved; one is assayed while the other serves as a replicate plate.

A further extension of this method to interface parallel HPLC separationwith inherently serial analytical methods such as mass spectrometry isnow described. FIG. 13 depicts, an array of capillary tubing (1) isinterfaced with a sample filled through-hole array (2). However theopposite end of the tubing array is splayed in such a manner so as toincrease the distance between consecutive rows (or columns) of the arraywhilst keeping the others intact. A series of spacers (3) through whichthe tubing is inserted to form the array provides structural integrity.The sample array is brought into contact and co-registered with thecapillary tubing array. Each capillary tube in the array is pre-filledwith porous gel suitable for chromatographic separation of the mixturescontained in the array through-holes. Pressurized carrier solvent isforced through the holes in the array and carries one sample into onecapillary tube. Lengths of the tubing in the array are chosen so as togive the desired separation efficiency for the components in themixtures analyzed. A fiber or thin tape runs just below and parallel toa row or column of the capillary tubing array. Lateral motion of thetubing array relative to the fiber brings the tubing ends in onecolumn/row in the array into contact with the fiber (or tape) (4). Fluidfrom each tube is transferred to discrete spatial locations along thefiber. After the fluid is transferred, the fiber is advanced through avacuum interface (5) and the fluid drops are sequentially presented tothe mass spectrometer (6) for analysis. After one set of drops isdeposited, the surface (e.g., nitrocellulose fiber) is advanced adistance sufficient for next set of drops to be deposited. Note thereare only two displacements required, the surface in one direction andthe array in the orthogonal direction.

With a combined time to make a mass spectral measurement and move thefiber of about 300 ms, a row of 100 drops is transported and analyzed in30 seconds. The entire 10,000-tube array is analyzed in 3000 seconds (50minutes). As shown in U.S. Pat. No. 6,005,664, stochastic sampling cansubstantially reduce the number of data points (by up to a factor of 10)needed to reconstruct a signal compared with equi-spaced sampling.Implementation of a stochastic sampling protocol could greatly reducethe analysis time.

Example 11 Optimization of Chemical Synthesis Conditions

Optimization of a chemical synthesis by changing one or more processparameters and recording the amount of material synthesized for a set ofreaction conditions. Modified process parameters include types ofreagents, reagent concentration, sequence of addition/mixing,temperature, and time.

A series of hydantoin compounds, pharmaceutical drugs useful fortreatment of epilepsy, are prepared using the new methods in solid-phasesynthesis. Each step in the synthesis process consists of reaction witha solvated reagent under a given set of time and temperature conditionsand then a wash to remove the excess (unreacted) reagents. Microspheresmade from resins having different functional groups (e.g., hydrogen,phenyl, methyl, benzyl, and s-butyl) and a protected amide group areeither purchased or synthesized. An array is manufactured with taperedholes such that as the beads are spread across the surface, only oneresin bead fits into each through-hole. Microspheres made from differentresins are mixed together and spread across the array in a dilutesolution such that all the holes are filled, each with a microspheremade from a different resin. Next, a mask is placed over the regulararray of through-holes and the amine groups of the exposed resin beadsare deprotected by exposure to a strong acid (e.g. trifluoroacetic acid)or base. After a wash step to remove the acid or base, the same resinsare exposed to one member from an isocyanate group library consisting ofdifferent chemical and structural moieties. Reaction between theisocyanates and amide groups from a urea with a variable moiety. Suchchemical units include hydrogen, butyl, allyl, 2-trifluorotoyl and4-methoxyphenyl. The reaction proceeds at an elevated temperature for acertain time after which the exposed holes are washed to removeunreacted reagents. This process is repeated with a new mask exposingsome of the original holes as well as new ones to expose thesemicrospheres to a new isocyanate library group. Upon completion, thehydantoins are screened against targets to find those potent compounds.Varying the reagents, their concentrations, the reagent sequences, thereaction temperature and reaction time provides a rapid and efficientmethod to optimize the synthetic rout.

Example 12 Selection of Phage Antibody Libraries (Multi-Chip Method)

A library of phage antibodies against a target antigen in a dense arrayof through-holes is screened. The protocol was adapted from (Winter,http://aximtLimt.uni-marburg.de/˜rek/AEPStart.html).

Through application of recombinant DNA technology a large (10⁹ to 10¹⁰)and diverse monoclonal antibody library is produced and stored as DNAplasmids in bacterial (E. coli) colonies. A single round of conventionalaffinity selection is performed in an immunotube coated with the targetantigen. After incubating, blocking, and washing, the specifically boundphage are eluted and used to reinfect additional E. coli.

Two 10,000-through-hole silicon arrays are fabricated. Both arraydevices are loaded with a solution of purified target antigen. The arraydevices are then incubated at 4° C. for approximately 12 hours, washedand filled with a blocking solution consisting of 4% dry milk powder inphosphate buffered saline (PBS). After one hour the blocking solution isremoved, the array devices are rinsed and refilled with a bufferedsolution of IPTG to induce phage expression in the bacteria.

A sufficiently dilute solution of the phage-infected bacteria is addedto a third 10,000 through-hole array such that there is an average of1-10 bacteria per through-hole. The resulting through-hole array,henceforth referred to as the Library Expression Chip, is incubateduntil the bacteria reach log phase. The Library Expression Chip isstacked between the two antigen coated through-hole arrays. The stack isthen incubated for two hours in a high humidity chamber to allow bindingof the phage antibodies to the antigen. The antigen through-hole arraysare washed to remove bacteria and unbound phage and separated from theLibrary Expression Chip.

The bound phage from one of the two antigen through-hole arrays,henceforth referred to as the Phage Inoculation Chip, is eluted byfilling the through-holes with a solution of 100 mM triethylamine,incubated for approximately 10 minutes, then neutralized by stackingonto another 10,000 through-hole array containing 2× Tris-HCl buffer.The eluted phage is used to inoculate uninfected bacteria contained in afourth 10,000 channel array. This array, henceforth referred to as thePositive Expression Chip, is grown to log phase and stored.

Meanwhile, the second antigen-phage chip, the Antibody Selection Chip,is analyzed for bound phage antibody via an indirect ELISA assay. Theprotocol is adapted from Current Protocols in Immunology, Supplement 15,pages 11.2.2-11.2.5. This protocol is an indirect ELISA to detectspecific antibodies. Other forms of ELISA assays, such as directcompetitive ELISA assays can be implemented in the through-hole arrayswith minor modifications to the procedure described in this example.

The Antibody Selection Chip is filled with a solution of the developingreagent, an-M13 conjugated to horseradish peroxidase in blockingsolution. After incubating for thirty minutes at room temperature, thearray is washed, blocked, and washed again. The array is then filledwith a solution of fluorescent substrate, and incubated for 30 minutesat room temperature. A fluorescent image is then collected every 5minutes for one hour. All positive through-holes are identified by anincrease in fluorescent intensity with time. Each correspondingbacterial culture is then extracted from the Positive Expression Chipand dispersed onto agar (containing ampicilin) in a separate cellculture well. These selected bacterial cultures constitute a source ofadditional phage antibody for subsequent analysis.

In an alternative method, the Library Expression plate, rather than thechip is replica plated. Only a single antigen through-hole array isexposed to the antibody library. After identifying positive interactionsin the Antigen Selection chip, the bacteria from the correspondingthrough-holes of the replicated Library Expression plate are diluted anddispersed across another 10,000 through-hole array. The assay isrepeated until to ensure that all selected bacterial colonies aremonocultures.

Example 13 Selection of Phage Antibodies (Single Plate Method)

A library of phage display antibodies is screened against a targetantigen using a single 10,000 channel array of through-holes. Byperforming the selection in a single array simplifies the screeningprocess, the phage must be reconstructed from their DNA, ecause theELISA assay renders selected phage incapable of reinfecting bacteria.

The target antigen is immobilized on the walls of the through-holes byfilling an array with antigen solution and incubating in theenvironmental chamber. Non-specific binding sites are blocked by washingthe array with blocking solution.

A solution of the phage-infected bacteria is added to all through-holesin the array such that there is an average of 1-10 bacteria perthrough-hole. The assay device is then incubated in a humidified chamberuntil the bacteria reach log phase. The assay device is submerged in asolution of IPTG for a period of time sufficient for diffusion of IPTGinto through-holes, but insufficient to permit bacteria to diffuse outof the array. The assay device is incubated to allow expression of phageantibody, and antibody-antigen binding. The expression of phage antibodyin the presence of the target antibody is counter to current methods inthe art, and can reduce the number of antibodies selected in the screen.

Next, the array is washed to remove bacteria, supernatant, and unboundphage from through-holes. Bound phage antibody is then detected by anindirect ELISA assay. After collecting a fluorescent image, the plate iswashed and filled with elution reagent to elute bound phage. Solutionsin through-holes that are identified corresponding to all positivethrough-holes by cherry picking to individual wells of a 96-well orhigher density microtiter plate. Phage DNA is then amplified by PCR andsequenced. The DNA sequences are used to identify the structure ofselected antibodies and to reproduce more phage antibody for furtherexperimentation.

Example 14 Protein Chip

One hundred thousand protein-binding probes are produced using phagedisplay technology according to the methods described in Sheets et al.,Proc. Natl. Acad. Sci. USA, 95:6157-6162, 1998, incorporated byreference in its entirety, such that each probe selectively binds aparticular human protein with high affinity and specificity. The probesare transferred into a platen, such that different protein-binding probein each of its through-holes. The platen has a TEFLON® surface toprevent wetting and protein absorption.

Tissue samples are homogenized, and the protein portions are extractedand equilibrated with the platen. The platen is washed to removenon-specifically bound molecules. The contents of each through-holes areanalyzed by heating the platen from ambient temperature to 100° C. overa two minute period while using Raman imaging to detect proteindesorption from the walls of the through-holes into the centers of thethrough-holes.

Example 15 Construction of an Array of Micro-HPLC Columns for RapidParallel Sample Separation and Purification

An array of twelve through-holes in a linear arrangement is machined ina block of material (e.g., metal, ceramic, or plastic). Thethrough-holes have a diameter of 250 μm, a total length of 20 mm, and acenter-to-center spacing of 9 millimeters. The internal diameter of thedistal end of the array of through-holes is increased and threaded toaccept standard 1/16″ outer diameter HPLC tubing using a standardnut-and-ferule compression fitting. A 2 cm long piece of 50 μm internaldiameter, 1/16″ outer diameter tubing is mated to each of thethrough-holes in the array through machined compression fittings. A1/16″ outer diameter stainless steel frit is placed inside thethrough-hole and is held in place with the compression fitting.Chromatography media is then packed into the each through-hole in thearray in the form of a slurry. The chromatography media is immobilizedwithin the through-hole due to the stainless steel frit at the distalend, and creates a micro-HPLC column. When pressurized, sample, wash,and elution buffers will flow through the chromatography media and thefrit, and elute from the through-holes through the mated 50 μm internaldiameter tubing at the distal end.

Samples are loaded onto the array of micro-HPLC columns by mating a bankof syringes to the micro-HPLC array as detailed in examples 2 and 3.Wash and elution buffers are flowed through the micro-column array usingthe same syringe bank. A flow rate of 1 to 20 μl of liquid per minute isused to wash and elute the samples from the micro-columns. The use ofnarrow bore tubing at the distal end of micro-HPLC columns results inthe liquid eluting from the column to form small droplets. The columneluate is collected in an array of wells (e.g., a microtiter plate). Theeluate is fractionated by collecting individual droplets as they elutefrom the micro-HPLC columns in different wells of the microtiter plate.Chemical and physical analysis (e.g., spectroscopy or spectrometry) ofthe samples can be performed on the samples within the wells.

Example 16 Fluidic Seals for Parallel HPLC in Microcapillary Channels

With reference to FIG. 16, an array of through-holes is machined in ablock of material (e.g., metal, ceramic, or plastic). The through-holeshave a diameter of less than 1 min and an aspect ratio greater than 10.The through-holes are chamfered to accept an o-ring gasket. A syringebank is fabricated with the same center-to-center spacing as thethrough-hole array. The syringe needles pass through a metal block thatis attached to the syringe bank holder by pneumatically actuated,spring-loaded pins. O-ring gaskets are placed onto the syringe needlesprotruding through the block. The syringes are loaded with fluid andinserted into the capillary tubes. The pins are pneumatically actuatedto bring the metal block in contact with the top surface of thecapillary tube block and press the o-ring gaskets into the hole chamferand around the syringe needle. This makes a leak tight seal between theneedle and capillary channel thus allowing the capillary channels to bepressurized by the syringes (FIG. 117). FIGS. 18 and 19 illustrate asimilar approach, except that the syringe bank in those figures is shownbolted to the capillary tube array to result in a rigid structure foreasy handling.

Example 17 Identifying a Ligand to a Biomolecular Target

The purpose of this experiment is to use low volume chromatography usingthrough-hole arrays to identify ligands in a chemical, biochemical orbiological mixture that bind to a biomolecular target, in this case aprotein. Two linear through-hole arrays are constructed such that eachthrough-hole holds 50 nl of liquid when filled. The exterior surfaces ofthe arrays are treated to be hydrophobic and the interiors are treatedto be hydrophilic. A center to center spacing of 9 mm between each ofthe through-holes is used and registration holes are includes to ensurealignment and co-registration of the through-holes upon stacking of thearrays using pins on a precision jig. A bank of syringes is used todispense 50 nl of target protein solution into the first array and 50 nlof different compound libraries into each through-hole of a secondarray. The arrays are stacked using a precision jig and the fluids areallowed to mix in each of the co-registered through-hole positions. Abank of syringes with compression fittings is filled with 1 ml ofeluant, followed by a small air bubble and the used to draw up the 100n1 of the protein-library reaction mixture. The reaction mixture is heldin the tip of the syringe and kept separate from the eluant reservoir bythe air gap. The syringe tips are the placed into the orifice of anarray of size exclusion columns and the compression fittings aretightened to ensure a seal. The samples are dispensed into the column,followed by the eluant. By monitoring the UV absorbance of the sampleexiting at least one column, one may determine when protein bound toligand is exiting the column. This protein is then recovered onto anarray of reverse phase columns by coupling the two column arrays. Theligands are removed from the protein by reverse phase chromatography,recovered, and analyzed to determine their identity.

Example 18 Protection of Through-Holes by Coating with Wax

Paraffin wax with a melting point of 54° C. is heated above its meltingpoint. A through-hole array is submerged in a thin layer of moltenpoly(ethylene glycol) wax with an average molecular weight of 1500. Thethrough-hole array and wax are cooled to cause the wax to harden. Excesswax is removed from the surface by scraping with a sharp blade and thenpolishing. The wax-filled array is then exposed for 10 seconds to vaporfrom a solution of(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane diluted 1:20in xylenes. The coating is then cured by baking the platen at 110° C.for 30 minutes while allowing the wax to melt and drip out of theplaten. Wax residues can be removed from the channel interiors bywashing with water. The platen is then rinsed for 10 seconds in sulfuricacid/hydrogen peroxide (2:1), and then with water, to remove residuesand ensure oxidation of the channel interiors. The resultingthrough-hole array has a hydrophobic exterior and a hydrophilicinterior.

Example 19 Photo-Initiation Method of Fiberglass Through-Hole ArrayProduction

A sheet of fiberglass filter such as that available from Millipore(Bedford, Mass.) is soaked in a polymerizable solution (e.g., a solutioncontaining methyl methacylate monomer and a photoinitiator such asbenzoin methyl ether) and placed between two quartz plates, each havingan array of dots that serve to mask the areas that will remain open andporous Polymerization of the monomer solution is effected byilluminating the photomasks with ultraviolet light.

Example 20 Application of an Assay to an Array of Through-Holes

A common assay used in drug discovery efforts is the cytochrome P450inhibition assay. Compounds that inhibit the activity of P450 enzymesare generally undesirable as pharmaceuticals as they have the potentialto cause serious side-effects and have the potential for negativereactions if taken in conjunction with other pharmaceuticals. A commonassay that is used to screen compounds that are potential candidates forbecoming pharmaceuticals is to incubate the compounds in a cellular,extra-cellular, or recombinant system than expresses P450 enzymes and toassay the activity of the enzymes in the presence of the candidatecompound. Such an assay could be modified for analysis in a massivelyparallel manner in an array of through-holes.

Four co-registered arrays of through-holes in which the through-holesare used. The enzyme or cellular compartment (e.g., microsomalcompartment of a liver cell) that expresses P450 enzymes can be loadedinto one array of through-holes such that each through-hole contains anequivalent volume and concentration of P450s. A known substrate for achosen P450 enzyme along with a source of NADPH (a source of energy) canbe loaded onto a second array of through-holes such that eachthrough-hole contains an equivalent volume and concentration ofsubstrate and NADPH. A third plate can be loaded with knownconcentrations (e.g., serial dilutions) of the compounds that are to beassayed along with positive and negative controls. An invention for therapid loading of multiple arrays of through-holes has been describedelsewhere in this document. The assay is started when these 3 arrays ofthrough-holes are brought in to contact with one another. As has beendescribed elsewhere in this document, by controlling the size, density,and surface chemistry of the through-holes and the arrays themselves thecontents of the 3 arrays can be made to rapidly mix with one anotherwhile inter-through-hole contamination between two or more adjacentthrough-holes can be avoided.

The stacked array of through-holes is then incubated at 37° C. in acontrolled humidity environment to allow the reaction to proceed. Bymaintaining a high humidity environment during this incubationevaporative loss from the through-holes can be minimized. After 30minutes a stop solution (e.g., organic solvent, urea, high salt solutionetc.) is added to the each through-hole by stacking a fourth arrayfilled with the stop solution with the assay arrays. The stop solutionworks to stop the assay by causing the P450 enzymes to denature and/orprecipitate out of solution. This precipitate can be pelleted bycentrifugation using an invention described elsewhere in this document.The top array of through-holes can then be removed from the others andused for analysis. In many assays, when the substrate is metabolized bythe P450 enzymes it is transformed from a non-fluorescent compound intoa fluorescent one. The amount of fluorescence will be directlyproportional to the amount of P450 activity. If the compound beingassayed inhibits the P450 enzyme, the less the metabolism that willoccur and less fluorescent material will be generated. The concentrationat which the P450 enzyme is inhibited by 50% (IC₅₀) can be determined ifmultiple concentrations of inhibitors were used in the assay.Fluorometric and/or mass spectrometric analysis of the samples can beperformed using inventions described elsewhere in this document.

Example 21 Washing Protein from a Surface of a Through-Hole Array

A through-hole array is prepared from silicon with afluoro-chloro-alkane coating on its surface and is treated with anoxidizing solution that renders the interiors of the holes hydrophilic.The array is dipped in water and frozen to −80° C., then quickly dippedinto a solution of the fluoropolymer FluoroPel® (Cytonix Corp.,Beltsville, Md.). The array is baked at 200° C. for 20 minutes, and thendipped into cell media containing 10% fetal calf serum. Dipping thethrough-hole array into the medium fills the holes in the array and wetsthe surface. By then dipping the through-hole array into perfluorooctaneand withdrawing slowly, the surface is cleaned of all aqueous media.

Example 22 DNA Sequencing

Fluorescently labeled DNA fragments are prepared by Sanger sequencingand arrayed in an array of 2500 through-holes in a platen of 0.5 mmthickness. This platen is then stacked on a second platen of 80 mmthickness containing a gel and inserted into an actively cooledelectrophoresis tank. As fluorescent oligos emerge from the column arraythey bind to a nitrocellulose membrane unwound from a spool. A computercontrols both the movement of the membrane and turns off the electricfield while the nitrocellulose membrane (in the form of a tape) ismoving. The membrane is then moved to cause exposure to a light sourceand CCD camera, analyzing the images and creating elution profiles foreach of the through-holes. This achieves reconstruction of the DNAsequence.

Example 23 Method of Manufacturing a Platen Using a Plurality of GroovedPlates

Platens having through-holes are to be manufactured from silicon plateshaving parallel grooves. See FIG. 20. The resulting array has 5000through-holes, wherein the through-holes are approximately square witheach side 0.25 mm in length and the platens are about 1 mm deep.

Nine inch silicon wafers (0.5 millimeter thickness) are used. Thecircular wafers are precisely cut to yield rectangular pieces (60 intotal) that are 55×160 millimeters (a total of three surfaces per waferare obtained). Grooves of 250 microns width and 250 micron depth arethen etched lengthwise into each piece of silicon. A total of 100grooves are etched into 50 of the silicon pieces and the distancebetween each groove is adjusted to about 250 microns. The remaining 10pieces of silicon are not etched. Chemical etching of silicon is aprocess known to those skilled in the art, and involves the masking theappropriate areas of silicon and treating with acid. The unmasked areasare then etched away in a controlled manner. A total of 25 millimetersfrom each side is not etched, providing a solid border to the finishedplatens.

Once the chemical etching of the surfaces is complete, the mask used inthe etching process is removed and the non-grooved surface of each pieceof silicon is sputter-coated with a thin layer of gold. The pieces ofsilicon are then stacked together in a jig having a flat surface with aright-angle bracket. Five pieces of silicon without grooves are firststacked, followed by the 50 grooved surfaces, and another 5 pieceswithout grooves. The entire jig containing the pieces of silicon ismoved to a press and pressure is applied to the stacked pieces ofsilicon. The assembly is heated and allowed to remain under elevatedpressure and temperature for about 16 hours. This treatment results inpermanent bonding of the platens into a single large piece of siliconwith 55×30×160 mm dimensions having 5000 through-holes (in a 100×50array) and a 25 mm solid border of silicon around the through-holes. Awire saw is used to cut the individual platens to a thickness of 0.5millimeter. The platens are cut by slicing in the plane orthogonal tothe through-holes. See FIG. 20. The wire used in the saw has a thicknessof 150 microns and as a result this amount of material is lost duringthe sawing process. Including the removal of uneven platens created ateither end, a total of 230 platens fitting the required specificationsare cut from this large piece of silicon.

Both surfaces of the platens are polished in a lapping machine to ensurea flat surface, resulting in an insignificant amount of silicon beingremoved. Finally, surface chemistry is applied to the platens to providethe finished product, having the desired physical characteristics.

Example 24 Storing and Screening a Nonoliter Volume Compound Library

Compounds are reformatted from 96 well plates into a through-hole arrayas follows: Compounds in 96 well plates are dissolved in DMSO to 100times the concentration that they willed be assayed at—for example, 100uM for a 1 uM final concentration in the assay. The total volume of DMSOsample solution in each well of the 96 well plate is 10 ul. Anadditional 90 ul of ethanol is mixed into each well and the samples areimmediately drawn into a syringe bank for dispensing. The automatedsyringe bank the dispensed 60 nl volume into each stack of athrough-hole array having the same footprint as a 96 well plate. Thisprocess is repeated with new 96-well plates until the through-holearrays are substantially full. The alcohol is then allowed to evaporate,leaving a residue of approximately 600 pl of DMSO dissolved compound ineach channel of each through-hole array. After storage of the compoundsfor the desired time under desiccation at −80° C., a through-hole arraycontaining the arrayed compounds is removed, brought to 10° C. so thatthe DMSO remains frozen and dipped into a beaker containing aqueousassay solution that has been chilled to 10° C. Removal of thethrough-hole array from the beaker under a humidified environmentresults in the through-holes of the chip being filled with the aqueousmedium. The through-hole array is warmed to ambient temperature to allowmixing of the solvents. A second through-hole array is uniformly filledwith a freshly prepared, chilled assay buffer containing an enzyme and afluorogenic substrate; in this case a Matrix Metalloprotease assay.Stacking of the two chips, warming to ambient temperature andfluorescent imaging at several time points over 30 minutes gives aprimary screen for enzyme inhibition.

Example 24 Selecting Membranes for Cell Culture

The invention provides methods for the growth of various cell types,including eukaryotic cells, including but not limited to adult stemcells, chondrocytes, embryonic stem cells, endothelial cells, epithelialcells, fibroblasts, hematopoietic cells, muscle cells, including cardiacmuscle cells of the heart, neurons, osteocytes). A schematic of thismethod is shown in FIG. 25. To facilitate the growth of these cells,porous membranes were assayed for their ability to support theattachment, survival, growth, or proliferation of an exemplary celltype, HEK 293 cells transfected with a PKCb-GFP expression vector (FIG.26). The cells were plated into wells of a 24-well plate (BD-Biocoat 24well) that contained inserts to be assayed. 3 μm pore inserts that wereuncoated or that were coated with fibronectin, laminin, and collagenwere tested, as were membranes fabricated from aluminum oxide having auniform capillary pore structure, ANOPORE tissue culture inserts, of0.2-μm pore size (Nunc Inc.). The membranes were incubated overnightwith 200,000 cells. Following this incubation, the ANOPORE membraneswere completely confluent (see FIG. 25). The laminin coated membrane wasabout 20% confluent. The other membranes were less than 10% confluent.Cells seeded at 75,000 per well on an Anopore insert continued to growuntil confluency at day 6 (see FIG. 27).

Example 25 Cell Chip Construction Methods

For cell chip construction, anodisc membranes, which are the membranesin the ANOPORE inserts, were used. First, membranes were placed on aglass slide and a 400 μm thick stainless steel platen with 150 μmdiameter pores was placed over the membrane. The device was placed in athe chamber of a Cytospin slide centrifuge and cells were added to thetop of the platen. After a twenty-four hour incubation the platen waswashed three times with cell culture medium. Although there were cellsadhering to the membrane, the cells were rounded and there were no firmattachments or spreading as seen in previous experiments using theanopore insert. With the inserts, the membrane was not placed on a solidsupport, but was suspended within the platen, which allow the membraneto be in contact with medium on both surfaces.

A double platen was constructed with a anodisc membrane in between theplatens as shown in FIG. 28. The bottom platen was pre-wetted by placinga drop of medium over the platen and moving a glass cover slipperpendicular to the platen over the surface to push the liquid into thepores. This was repeated on the top platen using a drop of cellsuspension at 1×10⁶ cells/mL. A pre-wetted membrane was then placedbetween the platens, and the platens were adjusted under a microscope sothat the pores of both platens were brought into register or alignment.The device was clamped and incubated overnight. Examination of themembrane after washing showed that the cells had a characteristicadherent morphology similar to that seen with the inserts (FIG. 28).Cells were examined every 24 hours and proliferated until confluent(FIG. 28).

Example 26 Array Spotting

To assess the potential of the cell-chip to be used with an arrayspotter for compound testing, single pin spotting was characterized(FIG. 29). FIG. 30 shows microspotting on gold platen using Hoechst dye.FIG. 36 shows microspotting of C12 resazurin to determine cellviability. C12 resazurin is a detection agent used to study cellularmetabolism. The reduction product of C12-resazurin is C12-resorufin,which exhibits enhanced cellular retention and detection relative to thereduction product of resazurin. Metabolically active cells reduce C12resazurin to C12 resorufin which fluoresces red. Hoechst stain wasincluded as a counter stain. After briefly spotting on the platen, thecell-chip was incubated for 15 minutes at 37° C., 5% CO₂. The chip wasexamined using fluorescence microscopy. Several wells in the area of thespot showed Hoechst staining and contained metabolically active cells.This area was about 300 uM in diameter, about 1.5 times the diameter ofthe pin (see FIG. 29). FIG. 36 shows an open array-based cell chip anddelivery of C12-resazurin to a single well on the array using a floatingpin.

The technology for microarray spotting allows the generation of highdensity microarrays by spotting cDNAs and/or oligonucleotides on a solidchip surface. In this report, the chip surface is modified providing forimproved performance of an ultra-high throughput cell-based screeningassay. This novel chip technology includes an ANODISC membrane selectedfor its ability to support cell attachment and viability and a microwellstainless steel platen.

Membranes with either 3 um or 0.2 μm pore diameter were tested for theirability to support PKCb-GFP cell attachment and growth. An ANODISC 0.2μm membranes was covered with a confluent layer of cells after anovernight incubation. In contrast, less than 10% confluency was presentwhen a polycarbonate membrane having a 3 μm pore size and treated withlaminin, collagen, fibronectin or untreated. These experiments suggestthat membranes having a smaller pore size have an enhanced ability tosupport cell attachment, differences in membrane material cannot beexcluded. Alternatively, difference in the fabrication material can notbe excluded.

When cells were cultured with a cell-chip consisting of a membrane on aglass slide covered by a stainless steel platen, the cells appearedround and did not attach well. In contrast, cells cultured on membranesin framed inserts (i.e., on a membrane between two platens) thatprovided contact with culture medium on both sides solved this problem.This configuration could can be adapted for use in a variety ofanalystical or culture systems. In one embodiment, a chip includingcells on a membrane is placed over another chip having a membranedesigned for protein or RNA attachment. Cells are lysed and the lysedcontents is spotted onto the second membrane by vacuum filtration fordot blot analysis.

These experiments described herein further demonstrated that a compoundcould be spotted directly onto the cell-chip and processed bymetabolically active cells with minimal diffusion on the platen. Inorder to decrease diffusion further and achieve high density spotting,the platen surface may be treated with a hydrophobic agent. Mineral oiland silicone coating dramatically limited diffusion and created a smallspot in studies carried out using membranes having 50 μm pore in a goldplaten (FIG. 31).

Because of the small well size, this novel technology provides for theculture and analysis of rare cell types and further provides ultra highthroughput single cell screening (uHTS). Single cell uHTS may have atransformational effect on antibody engineering as activated B cells maybe tested without fusion (variable regions amplified from positivecells).

The present invention overcomes limitations present in the prior art. Inparticular, using the cell-chip configuration described herein, livingcells can be analyzed using fluorescence markers for cell function.Further more, cultured cells may be lysed and the contents transferredthrough a membrane for further biochemical analysis, such as proteinanalysis or gene expression.

Example 27 Hoechst Staining of Cells

Cells were incubated overnight on a stainless steel cell-chip and thenwashed with medium. Hoechst was spotted on a first platen that was driedby blotting. The stainless steel platen (National Jet Company, LaVale,M.D.) tested was a 1-inch square, 400-μm thick with pores 150 μm indiameter. A second platen was attached to the first platen usingadhesive sealing film with the center cut out. The chip was placed in acytospin sample chamber with a small gasket between the platen and thetop of the chamber. No funnel was used. The results of this experimentare shown in FIG. 32. FIG. 33 shows an anopore membrane sandwichedbetween two stainless steel platens. PKCβ-GFP cells were added andincubated overnight.

Example 28 Tungsten Platens

A cell microarray prototype was constructed on a 200-μm thick Tungstenplaten. The platens (National Jet Company, LaVale, M.D.) had pores of300 μm in diameter and were attached with 4 screws. The prototype isshown in FIG. 34. An Anopore membrane of aluminum oxide was sandwichedbetween two tungsten platens (as shown in FIG. 34). PKCβ-GFP cells wereadded and incubated for 48 hours (FIG. 35). In this experiment, randomcell distribution was observed and the cells were more securely attachedto the substrate. Metal platens having biocompatibility may be used insuch methods. Metals that are not biocompatible may be coated with abiocompatible polymer (PEG) or metal.

Example 29 Confocal Images of Cultured Cells

FIG. 37 shows an open array-based cell chip with PKCβ-GFP transfectedcells, added at 5×10⁵/mL and incubated 37° C., 5% CO₂. Images wereacquired by confocal microscopy. FIG. 38 shows essentially that which isdepicted in FIG. 37, except here the platen is not shown.

Example 30 Rigid Materials May be Attached to the Porous Membrane

A platen of rigid material, such as metal or polystyrene having poresbetween 10-300 μm in diameter is attached to a porous membrane ormodified glass surface to form an array of microwells having a porousbottom (FIG. 39). Cells are added and allowed to adhere to the membraneovernight. Subsequently, one or more test compounds is added using amicro spotting pin. After incubation, high content image analysis isperformed. Membranes are processed to analyse protein, mRNA expression,or to detect changes in a biological function of interest. FIG. 40 showsa cross-section of an individual well depicted in FIG. 39. The presentinvention can be adapted as shown in FIG. 41. FIG. 41 depicts structuralenhancements to increase pressure on the member and/or gaskets to sealoff individual wells. Cell chip assays were carried out using thefollowing methods and materials.

Cell Culture Media

DMEM supplemented with 10% FCS and 0.22 ug/mL hygromycin was used tomaintain PKCb-GFP cells. In some experiments, DMEM without phenol redsupplemented with 10% FCS and 100 IU/mL penicillin, 100 ug/mLstreptomycin was used.

Cells

Human PKCBII cDNA (GenBank Accession No.:X07109) was isolated by PCRfrom reverse transcribed human spleen marathon-ready cDNA (Clontech) andsubcloned into the pCDNA3 vector containing a hygromycin resistant gene(Invitrogen). The gene was inserted downstream of sequences encoding agreen Xuorescent protein (ZsGFP) (Clontech). A stable cell lineexpressing GFP-PKC_II was obtained by transfecting pcDNA3/GFP-PKC_IIvector into human embryonic kidney (HEK) 293 cells (QBI, Montreal,Quebec, Canada) followed by selection with 600 μg/ml hygromycin B. Drugresistant colonies were picked and screened for PKCBII proteinexpression by immunoblotting using an anti-PKCBII antibody (Santa CruzBiotechnology, Santa Cruz, Calif.). The expression of GFP was tested byXuorescence microscopy. Positive clones were maintained in the growthmedium containing 300 μg/ml of hygromycin B. HEK293 cells transfectedwith GFP-tagged PKC-BII protein were maintained in DMEM containing 10%FBS, glutamine, antibiotics, and hygromycin B at 37° C. See, Ilyin etal., Methods 37: 280-288, 2005, which is hereby incorporated byreference.

Cell Culture on Membranes

Cell culture inserts from BD-Biocoat membranes were obtained in 3 uMpores size (BD Biosciences, San Jose, Calif.). The inserts werepre-coated with lamalin, collagen or fibronectin. Anopore inserts havinga 0.2 uM pore size (Nunc) membranes were also tested. Inserts wereplaced in a 24-well culture plate. Cell culture medium was added untilthe level reached the membrane. Cells were added to the inserts in 200uL of medium. Plates were incubated overnight at 37° C., 5% CO₂. Insubsequent experiments using membranes without framed inserts, anodisemembranes having 0.2 or 0.1 μm (Whatman, Clifton, N.J.) pores were used.

Platens

Stainless steel shims 400 μm thick were obtained and sent to NationalJet Company (LaVale, M.D.) for manufacture of a 10×10 platen of 150 uMdiameter holes spaced 50 μm apart using micro electro-dischargemachining. Each shim was cut to about 1 inch square with the platen inthe center.

Spotting Pins

An FP9 0.229 mm diameter floating tube pin with a volume delivery rangeof 5-15 nL (V&P Scientific, Inc., San Diego, Calif.) was used in allexperiments. Spotting solutions were made in an eppendorf tube. Thespotting pin was dipped in solution and then spotted on the platen bybriefly touching perpendicular to the platen.

Cell Viability Assay

C12-Resazurin (molecular probes) was diluted in DMEM 10% FCS w/o phenolred. Hoechst 33258 (molecular probes) was included to control forspotting efficiency. Solution was spotted onto platen on top of cells asdescribed above. The cell chamber was incubated at 37° C., 5% CO₂ for 15minutes. After incubation, the chamber was examined by fluorescencemicroscopy for Hoechst DNA staining. This area was then analyzed usingconfocal microscopy for conversion of C12-Resazurin to red-fluorescentresorufin by viable cells (Abs/Em 563/587).

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof. Allpatents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1-11. (canceled)
 12. A method of creating a chemical array, the methodcomprising: a) providing a platen having a plurality of through-holesand two opposing surfaces; b) applying a mask to one or both surfaces ofthe platen to block at least some of the through-holes, while leavingother through-holes open; c) exposing a surface of the platen to areagent so that the reagent enters at least one of the openthrough-holes; and d) repeating steps b) and c) with at least onedifferent mask and at least one different reagent to create a chemicalarray.
 13. The method of claim 12, wherein the mask is made of apolymer, an elastomer, paper, glass, or a semiconductor material. 14.The method of claim 12, wherein the mask comprises mechanical valves,pin arrays, or gas jets.
 15. The method of claim 12, wherein theapplying step forms a hermetic seal between the mask and the platen. 16.The method of claim 12, wherein the reagent is a liquid, a gas, a solid,a powder, a gel, a solution, a suspension, a cell culture, a viruspreparation, or electromagnetic radiation.
 17. The method of claim 12,wherein the mask is translated to expose different through-holes. 18.The method of claim 12, wherein the mask has co-registration pins andholes such that alignment of pins and holes in the mask register withthe throughholes in the platen.
 19. The method of claim 12, wherein themask comprises a flexible material.
 20. The method of claim 19, whereinmultiple masks are part of a flexible tape, and the multiple masks areregistered with the through-holes of the platen by advancing the tape.21. An array created by the method of claim
 12. 22-50. (canceled)
 51. Adevice for filling or draining through-holes in a platen having aplurality of through-holes, the device comprising: a) a holder adaptedto accept the platen; b) a nozzle having an aperture of a suitable sizeto inject a sample into a single through-hole in said platen; and c) avalve that controls a flow of a sample through said nozzle, wherein theholder and nozzle can move with respect to each other.
 52. The device ofclaim 51, wherein the nozzle is positioned so as to contact the platen.53. The device of claim 51, further comprising a microplate positionedto receive samples from the platen.
 54. The device of claim 51, furthercomprising a computer that controls the valve and controls the positionsof the holder and nozzle relative to each other.
 55. The device of claim53, further comprising a computer that controls the valve and controlsthe positions of the microplate and holder relative to each other. 56.The method of claim 53, wherein the microplate, the holder, and thenozzle can be moved independently of each other in at least twodimensions.
 57. The method of claim 51, wherein the nozzle is held in asingle position and the holder and nozzle can be moved independently ofeach other in at least two dimensions. 58-127. (canceled)